Acidophilic fusarium oxysporum strains, methods of their production and methods of their use

ABSTRACT

The present invention provides isolated acidophilic  Fusarium oxysporum  strains, such as MK7, and their progeny, compositions comprising such strains and their progeny, methods of producing such strains and their progeny, and methods of using such strains and their progeny.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application No.62/020,607, filed on Jul. 3, 2014, and U.S. provisional application62/061,076, filed on Oct. 7, 2014, each of which is hereby incorporatedby reference in its entirety for all purposes.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety; A computer readableformat copy of the Sequence Listing (filename:MONT_(—)151_(—)03US_ST25.txt, date recorded: Jul. 1, 2015, file size 2kilobytes).

TECHNICAL FIELD

This application relates to novel isolated fungal strains of acidophilicFusarium oxysporum, (e.g., MK7), their progeny, methods of producingsuch isolated fungal strains and their progeny, and methods of usingsuch isolated fungal strains and their progeny to produce usefulprocesses and products. For example, the isolated fungal strains andtheir progeny are useful for converting ligno-cellulosic feedstocks,algal biomass, and glycerol into energy-rich metabolites for bioenergy,for producing enzymes, antibiotics, specifically fatty acids and lipids(e.g. waxes) for commercial applications, dewaxing straw and forneutralizing acid and binding heavy metals.

BACKGROUND

Fusarium (synonyms: Fusisporium, Pseudofusarium, Sporotrichella) is alarge genus of filamentous fungi widely distributed in soil and inassociation with plants. Most species are harmless saprobes and arerelatively abundant members of the soil microbial community. Somespecies produce mycotoxins in cereal crops that can affect human andanimal health if they enter the food chain. The main toxins produced bythese Fusarium species are fumonisins and trichothecenes.

Fusarium strains isolated from nature have proven useful to mankind in awide variety of technologies, including for use as food and for theproduction of antibiotics. The need still exists for new isolates ofFusarium for use in new and existing applications. The present inventionprovides a unique isolated strain of Fusarium and its progeny which areuseful for a number of different purposes, including but not limited tobioenergy production, as biolubricants, for biorecovery of preciousmetals by biosorption, for remediation of mine waste, for dewaxing ofwheat straw, for degradation of algal biomass, and glycerol containingwaste products, and for antibiotic, siderphore, and plasticizerproduction.

SUMMARY

The present invention provides isolated fungal strains of acidophilicFusarium oxysporum, such as the isolated strain designated as MK7, whichhas been deposited as ATCC Deposit No. PTA-10698, and/or its progeny.“Progeny” as used herein refers to any and all descendants by lineagewhich originate from the isolated strain no matter however or whereverproduced. Included within the definition of “progeny” as used herein areany and all mutants of the isolated/deposited strain and its progeny,wherein such mutants have all of the physiological and morphologicalcharacteristics of the isolated/deposited strain and its progeny.

In one embodiment, the present invention provides an isolatedligno-cellulose degrading acidophilic fungal strain of Fusariumoxysporum or its progeny, wherein the isolated fungal strain of Fusariumoxysporum has at least the following identifying characteristics:

-   -   a) the isolated strain is acidophilic and can grow at a pH        ranging from about 0.7 to about 7.5; and    -   b) ability to produce lipids from ligno-cellulosic feedstocks,        carbon containing waste products, carbohydrates, or a        combination thereof under aerobic or substantially aerobic        conditions; In one embodiment, the isolated strain of the        present invention further comprises one or more the following        additional identifying characteristics:    -   c) ability to produce ethanol and/or hydrogen from        ligno-cellulosic feedstocks, carbon containing waste products,        carbohydrates, or a combination thereof under anaerobic or        substantially anaerobic conditions;    -   d) ability to tolerate Mn concentrations of up to 25 mM, As        concentrations up to 250 mM or 300 mM, and Hg concentrations of        up to 100 mM;    -   e) ability to dewax wheat straw and other plant material;    -   f) ability to produce lipids from algal feedstocks and from        waste generated during biofuel production (e.g. processed algal        biomass, glycerol) under aerobic or substantially aerobic        conditions;    -   g) ability to produce ethanol and/or hydrogen from algal        feedstocks and from waste generated during biofuel production        (e.g. processed algal biomass, glycerol) under anaerobic or        substantially anaerobic conditions;    -   h) ability to produce Diisooctyl phthalate (DIOP);    -   i) ability to produce Hexanedioic acid, mono(2-ethylhexyl)        ester; and    -   j) comprising an 18S rRNA and ITS region DNA sequence that        shares at least 98% identity to SEQ ID NO. 1.

In some embodiments, the ligno-cellulosic feedstocks utilized by theisolated strain and its progeny are selected from the group consistingof agricultural crop residues (e.g., wheat straw, barley straw, ricestraw, small grain straw, corn stover, corn fibers (e.g., corn fiber gum(CFG), distillers dried grains (DDG), corn gluten meal (CGM)), switchgrass, hay-alfalfa, sugarcane bagasse), non-agricultural biomass (e.g.,algal mats, urban tree residue), forest products and industry residues(e.g., softwood first/secondary mill residue, hard softwoodfirst/secondary mill residue, recycled paper pulp sludge),ligno-cellulosic containing waste (e.g., newsprint, waste paper, brewinggrains, used rubber tire (UTR), municipal organic waste, yard waste,clinical organic waste, and waste generated during the production ofbiofuels (e.g. processed algal biomass, glycerol), and combinationthereof. In some embodiments, the carbohydrates are selected from thegroup consisting of monosaccharides, disaccharides, oligosaccharides,polysaccharides, and mixture thereof.

In some embodiments, the said isolated fungal strain and its progeny cangrow and/or multiply at a pH ranging from about 0.7 to about 7.0. Forexample, the strains and progeny of the instant invention are able togrown and/or multiply at a pH of at least about 7.0, at least about 6.5,at least about 6.0, at least about 5.5, at least about 5.0, at leastabout 4.5, at least about 4.0, at least about 3.5, at least about 3.0,at least about 2.5, at least about 2.0, at least about 1.9, at leastabout 1.8, at least about 1.6, at least about 1.4, at least about 1.2,at least about 1.0, at least about 0.9, at least about 0.8, at leastabout 0.75, at least about 0.7, at least about 0.65, at least about 0.6,or at least about 0.55.

In some embodiments, the isolated fungal strain and its progeny areresistant to high concentrations of other metals selected from the groupconsisting Ag, Zn, Fe, Al, Be, Pb, Cu, Cr, Ni, Cd, Co, Ni, Pd, Pt, U,Th, Mo, Sn, Ti, As, Au, Se, Sb and Hg.

The isolated Fusarium oxysporum strain and its progeny of the presentinvention can be cultured in the absence of antibiotics with little orno contamination by other organisms, wherein said other organisms can beselected from the group consisting of other bacterial strains, genus orspecies, other fungi (e.g., yeasts, molds), algae, viruses, plants,insects, and any combination thereof.

The present invention also provides a biologically pure culture havingone or more of the identifying characteristics of the isolated fungalstrain of Fusarium oxysporum or its progeny as described herein. In oneembodiment, the biologically pure culture comprises the isolated fungalstrain of Fusarium oxysporum, designated as MK7, which has beendeposited as ATCC Deposit No. PTA-10698, and/or progeny thereof. In oneembodiment, said isolated fungal strain and/or progeny thereof is in theform of conidia, pycnidia, chlamydospores, fragments of hyphae, or anyand all combinations thereof.

The present invention further provides a composition comprising anisolated fungal strain of Fusarium oxysporum and/or its progeny havingat least one or more of the identifying characteristics as describedherein. In one embodiment, the isolated fungal strain is MK7 and/or theprogeny thereof. In one embodiment, said compositions comprise theisolated fungal strain and/or progeny thereof in the form of conidia,pycnidia, chlamydospores, fragments of hyphae, or any and allcombinations thereof. In some embodiments, the composition furthercomprises one or more components selected from the group consisting of amedium that supports growth of the fungal strain, an acidificationmaterial, a manganese donor, a nutrient addition, and any and allmixtures thereof. In one embodiment, the medium is a solid. In anotherembodiment, the medium is a liquid.

The present invention also provides methods of producing useful productsusing said isolated fungus stain and/or its progeny, comprising:

a) making a mixture of one or more said isolated fungal strains and/orits progeny with a feedstock material selected from the group consistingof ligno-cellulosic feedstocks, carbon containing agricultural andmunicipal waste products, carbohydrates, sugar monomers, yeast extract,casamino acids, and a combination thereof in a container, wherein thematerial can support the growth of said isolated fungal strain; andb) growing said isolated fungal strain in said mixture to produce one ormore useful products.

In one embodiment, the mixture is under aerobic conditions orsubstantially aerobic conditions. In another embodiment, the mixture isunder anaerobic conditions or substantially anaerobic conditions.

In one embodiment, said useful products are one or more energy-richmetabolites. For example, the useful products produced using the fungiand methods of the present invention include but are not limited tobiofuels, and/or precursors of biofuels. In one embodiment, the mixtureis under aerobic conditions or substantially aerobic conditions, andsaid energy-rich metabolites are essentially lipids, wherein the lipidscan be extracted from the biomass of the isolated fungal strains and/orits progeny. In one embodiment, said lipids are essentially fatty acids.In one embodiment, said fatty acids are essentially unsaturated fattyacids and/or saturated fatty acids. In one embodiment, said unsaturatedfatty acids are selected from the group consisting of oleic acid (18:1),α-linolenic acid (18:3), eicosenoic acid (20:1), and a combinationthereof. In one embodiment, said saturated fatty acids are selected fromthe group consisting of palitic acids (16:0), stearic acids (18:0),arachidic acid (20:0), behenic acid (22:0), and any and all combinationsthereof. In one embodiment, lipids are extracted and used to producebiolubricants. In one embodiment, the mixture in under anaerobicconditions or substantially anaerobic conditions, and said energy-richmetabolites are essentially alcohols, and/or hydrogen. In someembodiments, said alcohol is ethanol, butanol and/or isobutanol.

In one embodiment, said useful products are naphthazarinoid pigments. Ina further embodiment, said naphthazarinoid pigments are harvested andused to produce pesticides selected from the group consisting ofantibiotics, fungicides, herbicides, yeasticides, and insecticides.

In one embodiment, said useful products are siderophores.

In one embodiment, said useful products are plasticizers, e.g.,Diisooctyl phthalate (DIOP, CAS 117-81-7) and/or Hexanedioic acid,mono(2-ethylhexyl) ester (or 2-Ethylhexyl hydrogen adipate, CAS4337-65-9).

Compared to the methods previously known in the art, the presentinvention is unique at least in that the production process as describedherewith is very simple in execution, whereby feedstocks are converteddirectly into lipids at high yield under acidic condition. In oneembodiment, ligno-cellulosic feedstocks are selected from the groupconsisting of agricultural crop residues (e.g., wheat straw, barleystraw, rice straw, small grain straw, corn stover, corn fibers (e.g.,corn fiber gum (CFG), distillers dried grains (DDG), corn gluten meal(CGM)), switch grass, hay-alfalfa, sugarcane bagasse), non-agriculturalbiomass (e.g., algal mats, urban tree residue), forest products andindustry residues (e.g., softwood first/secondary mill residue, hardsoftwood first/secondary mill residue, recycled paper pulp sludge),ligno-cellulosic containing waste (e.g., newsprint, waste paper, brewinggrains, used rubber tire (UTR), municipal organic waste, yard waste,clinical organic waste, and waste generated during the production ofbiofuels (e.g. processed algal biomass, glycerol), or any and allcombinations thereof. In one embodiment, said carbohydrates are selectedfrom the group consisting of monosaccharides, disaccharides,oligosaccharides, polysaccharides, and a combination thereof.

In one embodiment, said sugar monomers are selected from the groupconsisting of trioses, tetroses, pentoses, hexoses, heptoses, et al.,and any and all combinations thereof. In one embodiment, said pentosesare selected from the group consisting of ribulose, xylulose, ribose,arabinose, xylose, lyxose, deoxyribose, and any and all combinationsthereof. In one embodiment, said hexoses are selected from the groupconsisting of allose, altrose, glucose, mannose, glucose, idose,galactose, talose, psicose, fructose, sorbose, tagatose, and any and allcombinations thereof.

In one embodiment, said mixture further comprises at least one componentselected from the group consisting of acidification materials, manganesedonors, nutrient additions, pH buffering materials, and any and allcombinations thereof.

In some embodiments, said mixture has an initial pH from about 0.5 toabout 3.0. In other embodiments, said mixture has an initial pH fromabout 3.0 to about 7.0. The pH is determined based on the products. Forexample, high lipid production takes place over a the pH range 2.0-7.0,ethanol production takes place between pH 3-4.5 and H₂ production takesplace over a range of 2.0-7.0.

The present invention also provides methods of producing biofuel orprecursor of biofuel using one or more said isolated fungus stainsand/or the progeny thereof, said methods comprising:

a) making a mixture of one or more said isolated fungal strains and/orits progeny with a feedstock material selected from the group consistingof ligno-cellulosic feedstocks, carbon containing waste products,carbohydrates, sugar monomers, and any and all combinations thereof in acontainer, wherein the material can support the growth of said isolatedfungal strains;

b) growing said isolated fungal strain in said mixture to produce one ormore types of energy-rich metabolites as biofuel and/or precursors ofbiofuel;

c) harvesting said energy-rich metabolites; and

d) optionally, producing biofuel from said biofuel precursors.

In one embodiment, said biofuel is biodiesel. In another embodiment,said biofuel is bioalcohols. In still another embodiment, said biofuelis biogas.

The present invention also provides methods of pretreating cellulosicwaste and simultaneous conversion to energy-rich metabolites using oneor more isolated fungus stains and/or its progeny thereof, said methodscomprising:

a) making a mixture of one or more said isolated fungal strains and/orits progeny with cellulosic waste in a container; and

b) growing said isolated fungal strain and/or its progeny in saidmixture, wherein the cellulosic waste is degenerated and energy-richmetabolites are simultaneously produced.

In one embodiment, the cellulosic waste can support the growth of saidisolated fungal strains and/or its progeny. In another embodiment,additional compounds that are necessary for the growth of said isolatedfungal strains and/or its progeny are also added into the mixture,wherein the additional compounds are not provided by the cellulosicwaste. Such compounds include, but are not limited to, macronutrients,micronutrients, and combination thereof. In still another embodiment,compounds that can facilitate the pretreatment can be also added intothe mixture. Such compounds include, but are not limited toacidification materials, manganese donors, nutrients, pH bufferingmaterials.

The present invention also provides methods of detoxifying fluid wastecontaining metals and/or recovering metals from said fluid waste throughbiosorption using one or more said isolated fungus stains and/or itsprogeny thereof, comprising making a mixture of one or more saidisolated fungal strains and/or its progeny with the waste materialcontaining one or more types of precious metals, wherein the fluid wasteis detoxified and/or the metals are recovered through biosorption.

In one embodiment, the fluid waste can support the growth of saidisolated fungal strains and/or its progeny. In another embodiment,additional compounds that are necessary for the growth of said isolatedfungal strains and/or its progeny are also added into the mixture,wherein the additional compounds are not provided by the fluid waste.Such compounds include, but are not limited to, macronutrients,micronutrients, and combination thereof. In one embodiment, thebiosorption is through siderophores produced in the isolated fungalstrain. In one embodiment, the waste material is mining or industrialeffluents. In one embodiment, the metal is selected from the groupconsisting of Mn, Ag, Zn, Fe, Al, Be, Pb, Cu, Cr, Ni, Cd, Co, Ni, Pd,Pt, U, Th, Mo, Sn, Ti, As, Au, Hg and any and all combinations thereof.The total concentration of the metal ion(s), or the concentration of acertain metal ion in the fluid is at least 0.1 ppm, 1 ppm, or 10 ppm, or100 ppm, or 1000 ppm, or 10000 ppm, or 10000 ppm, or 100000 ppm, byweight. For example, the concentration of a certain metal ion in thefluid is higher than the requirement in the Federal Hazard Waste CodesEPA D004-EPA D013.

The present invention also provides methods of neutralizing pH of acidicfluids using one or more said isolated fungus stains and/or its progenythereof, comprising making a mixture of one or more said isolated fungalstrains with the acidic fluid, wherein the pH of the acidic fluids isneutralized. In one embodiment, the acidic fluid is acid mine drainage.In one embodiment, the acidic fluids can support the growth of saidisolated fungal strains and/or its progeny. In another embodiment,additional compounds that are necessary for the growth of said isolatedfungal strains and/or its progeny are also added into the mixture,wherein the additional compounds are not provided by the acidic fluids.Such compounds include, but are not limited to, carbon containingfeedstocks, macronutrients, micronutrients, and any and all combinationsthereof.

The present invention also provides acid-tolerant enzymes or portions ofthe acid-tolerant enzymes, and nucleic acids encoding said enzymes,portions of the acid-tolerant enzymes, or variants thereof, wherein suchenzymes are produced by the isolated strain and/or its progeny.

The present invention also provides mutated or recombinant fungalstrains derived from the fungal strain and/or its progeny as provided bythe present invention.

The present invention also provides methods of using isolated fungalstrains of acidophilic Fusarium oxysporum, such as the isolated straindesignated as MK7, to directly convert a wide range of feedstocks suchas wheat straw, corn stover, and industrial by-products (e.g. molasses,glycerol) to valuable lipid products such as Omega-7 fatty acids and/orhigh-melting temperature waxes.

The present invention also provides simple, novel and cost-effectiveprocesses for converting lignocellulosic and other waste feedstocks tohigh value lipids using isolated fungal strains of acidophilic Fusariumoxysporum, such as the isolated strain designated as MK7, which iscapable of withstanding extreme acidic conditions and producing powerfulenzymes for degrading cellulose, lignin and hemicellulose. The organismaccumulates high concentrations of valuable lipids in a cost-effective“one-step” process.

In some embodiments, the present invention teaches a method forproducing an energy-rich substrate, said method comprising contacting acarbon source with an isolated Fusarium oxysporum strain to form amixture, incubating said mixture for a period of time, and obtaining theenergy-rich substrate following such contact.

In some embodiments, the present invention teaches an MK7 Fusariumoxysporum strain, for which a representative sample has been depositedas ATCC Accession Deposit No. PTA-10698.

In some embodiments, the present invention methods of producingenergy-rich substrates using a Fusarium oxysporum strain with an rRNAsequence as disclosed in SEQ ID No.: 1.

In some embodiments, the application teaches that the carbon source usedin the methods of the present invention is a biomass product.

In some embodiments, the application teaches that the carbon source usedin the methods of the present invention is a cellulosic biomass product.

In some embodiments, the application teaches that the carbon source usedin the methods of the present invention is a ligno-cellulosic feedstockor lingo-cellulosic waste.

In some embodiments, the application teaches that the carbon source usedin the methods of the present invention is a carbohydrate.

In some embodiments, the present invention teaches methods wherein theFusarium oxysporum strain and the carbon source are incubated inanaerobic or microaerobic conditions.

In some embodiments, the methods of the present invention produce anenergy-rich substrate, wherein said energy-rich substrate is selectedfrom the group consisting of ethanol and hydrogen gas.

In some embodiments, the present invention teaches a pretreatment step,wherein said pretreatment step is selected from the group consisting of:reducing the pH of the carbon source and Fusarium oxysporum mixture,adding manganese to the carbon source and Fusarium oxysporum mixture,and adding a nutrient to the carbon source and Fusarium oxysporummixture before the incubation step.

In some embodiments, the energy-rich substrate produced by the methodsof the present invention is a lipid, ethanol, and/or hydrogen.

In some embodiments, the energy-rich substrate produced by the methodsof the present invention is a lipid.

In some embodiments, the present invention teaches a method forproducing one or more energy-rich metabolites using a Fusarium oxysporumstrain, said method comprising the steps of: a) making a mixture of aFusarium oxysporum MK7 strain, and/or its progeny, with a feedstockmaterial selected from the group consisting of ligno-cellulosicfeedstocks, carbon containing waste products, carbohydrates, and acombination thereof in a container, wherein the material can support thegrowth of said MK7 strain and/or its progeny; b) growing said MK7 strainin said mixture to produce one or more energy-rich metabolites; and c)optionally, isolating said one or more energy-rich metabolites from themixture; wherein a representative sample of said MK7 strain has beendeposited as ATCC Accession Deposit No. PTA-10698.

In some embodiments, the present invention teaches methods of producingenergy-rich substrates from wheat straw feedstock.

In some embodiments, the energy-rich substrate produced by the methodsof the present invention is ethanol.

In some embodiments, the energy-rich substrate produced by the methodsof the present invention is hydrogen gas.

In some embodiments, the energy-rich substrate produced by the methodsof the present invention is a fatty acid methyl ester (FAME).

In some embodiments, the present invention teaches FAME selected fromthe group consisting of methyl tetradecanoate, methyl hexadec-9-enoate,hexadecanoic acid, methyl ester, oleic acid, linoleic acid, conjugatedlinoleics, linolinec acid, methyl 11-octadecenoate, Octadecanoic acid,methyl ester, eicosanoic acid, methyl ester, docosanoic acid, methylester, and tetracosanoic acid, methyl ester.

In some embodiments, the application teaches that the FAME produced bythe methods of the present invention is an Omega-7 vaccenic acid.

In some embodiments, the present invention teaches that the mixture ofFusarium and energy source (e.g., carbon source or feedstock) isincubated in anaerobic or microaerobic conditions.

In some embodiments, the methods of the present invention comprise apretreatment step, wherein said pretreatment step is selected from thegroup consisting of: reducing the pH of the mixture, adding manganese tothe mixture, and adding a nutrient to the mixture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an inventive process that utilizes Fusarium oxysporumstrain MK7, a novel acidophilic fungus, and/or its progeny to directlyconvert ligno-cellulosic feedstocks, 5 and 6 carbon sugars, and algalbiomass to energy-rich substrates including lipids, ethanol andhydrogen. Additionally, the fungus can be used to produce enzymes andantibiotics for commercial applications. Additionally, the fungus iscapable of neutralizing acid and binding heavy metals for theremediation of acid mine drainage.

FIG. 2 depicts fungal biomass after 7 d of growth on wheat straw at pH2.5 under aerobic conditions. This fungal mat is ready for lipidextraction.

FIG. 3 depicts total lipid production by Fusarium strain MK7 and/or itsprogeny as a fraction of initial glucose or wheat straw. Wheat strawpercentage (4% or 8%) indicates initial mass of wheat straw to volume ofwater. Error bars show standard deviations of three experiments.

FIG. 4 depicts optical microscopy (left) of Fusarium strain MK7 and/orits progeny cultured on wheat straw at pH 2.5 after 10 days. Identicalview obtained using fluorescent microscopy of Nile Red stained cells(right) clearly reveal significant lipid production (Nile Redspecifically stains lipids, Cooksey et al., 1987).

FIG. 5 depicts fatty acid profile for Fusarium strain MK7 and/or itsprogeny cultured on wheat straw at pH 2.5 after 10 days.

FIG. 6 depicts ethanol yields in grams of ethanol produced per gram ofdry wheat straw feedstock or glucose.

FIG. 7 depicts lipid profiles produced by strain MK7.

DETAILED DESCRIPTION Definition

As used herein, the verb “comprise” as is used in this description andin the claims and its conjugations are used in its non-limiting sense tomean that items following the word are included, but items notspecifically mentioned are not excluded. In addition, reference to anelement by the indefinite article “a” or “an” does not exclude thepossibility that more than one of the elements are present, unless thecontext clearly requires that there is one and only one of the elements.The indefinite article “a” or “an” thus usually means “at least one”.

As used herein, the term “derived from” refers to the origin or source,and may include naturally occurring, recombinant, unpurified, orpurified molecules. The proteins and molecules of the present inventionmay be derived from influenza or non-influenza molecules.

As used herein, the term “microaerobic” refers to an environment wherethe concentration of oxygen is less than the concentration of oxygen inair.

As used herein, the term “chimeric protein” refers a constructs thatlinks at least two heterologous proteins into a single macromolecule(fusion protein).

As used herein, the term “nucleic acid” refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides, or analogs thereof. This term refers to theprimary structure of the molecule, and thus includes double- andsingle-stranded DNA, as well as double- and single-stranded RNA. It alsoincludes modified nucleic acids such as methylated and/or capped nucleicacids, nucleic acids containing modified bases, backbone modifications,and the like. The terms “nucleic acid” and “nucleotide sequence” areused interchangeably.

As used herein, the term “gene” refers to a nucleic acid or portion of anucleic acid comprising a sequence that encodes a protein. It isunderstood in the art that a gene also comprises non-coding sequences,such as 5′ and 3′ flanking sequences (such as promoters, enhancers,repressors, and other regulatory sequences) as well as introns.

As used herein, the terms “polynucleotide”, “polynucleotide sequence”,“nucleic acid sequence”, “nucleic acid fragment”, and “isolated nucleicacid fragment” are used interchangeably herein. These terms encompassnucleotide sequences and the like. A polynucleotide may be a polymer ofRNA or DNA that is single- or double-stranded, that optionally containssynthetic, non-natural or altered nucleotide bases. A polynucleotide inthe form of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides(usually found in their 5′-monophosphate form) are referred to by asingle letter designation as follows: “A” for adenylate ordeoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate ordeoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate,“T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines(C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N”for any nucleotide.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. These terms also include proteins that are post-translationallymodified through reactions that include glycosylation, acetylation andphosphorylation.

As used herein, the term “homologous” or “homologue” or “ortholog” isknown in the art and refers to related sequences that share a commonancestor or family member and are determined based on the degree ofsequence identity. These terms describe the relationship between a genefound in one species, subspecies, variety, cultivar or strain and thecorresponding or equivalent gene in another species, subspecies,variety, cultivar or strain. For purposes of this invention homologoussequences are compared. “Homologous sequences” or “homologues” or“orthologs” are thought, believed, or known to be functionally related.A functional relationship may be indicated in any one of a number ofways, including, but not limited to: (a) degree of sequence identityand/or (b) the same or similar biological function. Preferably, both (a)and (b) are indicated. The degree of sequence identity may vary, but inone embodiment, is at least 50% (when using standard sequence alignmentprograms known in the art), at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least 98.5%, or at least about 99%, or at least 99.5%, or at least99.8%, or at least 99.9%. Homology can be determined using softwareprograms readily available in the art, such as those discussed inCurrent Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987) Supplement 30, section 7.718, Table 7.71. Some alignment programsare MacVector (Oxford Molecular Ltd, Oxford, U.K.) and ALIGN Plus(Scientific and Educational Software, Pennsylvania). Another alignmentprogram is Sequencher (Gene Codes, Ann Arbor, Mich.), using defaultparameters.

As used herein, the term “nucleotide change” refers to, e.g., nucleotidesubstitution, deletion, and/or insertion, as is well understood in theart. For example, mutations can be those containing alterations thatproduce silent substitutions, additions, or deletions, but do not alterthe properties or activities of the encoded protein or how the proteinsare made.

As used herein, the term “protein modification” refers to, e.g., aminoacid substitution, amino acid modification, deletion, and/or insertion,as is well understood in the art.

As used herein, the term “derived from” refers to the origin or source,and may include naturally occurring, recombinant, unpurified, orpurified molecules. A nucleic acid or an amino acid derived from anorigin or source may have all kinds of nucleotide changes or proteinmodification as defined above. A fungi derived from a specific, isolatedfungal strain and/or its progeny may comprise certain mutations butstill retain one, two, or more, or all of the distinguishingmorphological and physiological characteristics of the isolated fungi orits progeny from which they were derived.

As used herein, the term “acidophilic” refers to that an organism whoseoptimal growth conditions are under acidic conditions.

As used herein, the term “agent”, as used herein, means a biological orchemical compound such as a simple or complex organic or inorganicmolecule, a peptide, a protein or an oligonucleotide that modulates thefunction of a nucleic acid or polypeptide. A vast array of compounds canbe synthesized, for example oligomers, such as oligopeptides andoligonucleotides, and synthetic organic and inorganic compounds based onvarious core structures, and these are also included in the term“agent”. In addition, various natural sources can provide compounds forscreening, such as plant or animal extracts, and the like. Compounds canbe tested singly or in combination with one another.

As used herein, the term “at least a portion” of a nucleic acid orpolypeptide means a portion having the minimal size characteristics ofsuch sequences, or any larger fragment of the full length molecule, upto and including the full length molecule. For example, a portion of anucleic acid may be 12 nucleotides, 13 nucleotides, 14 nucleotides, 15nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19nucleotides, 20 nucleotides, 22 nucleotides, 24 nucleotides, 26nucleotides, 28 nucleotides, 30 nucleotides, 32 nucleotides, 34nucleotides, 36 nucleotides, 38 nucleotides, 40 nucleotides, 45nucleotides, 50 nucleotides, 55 nucleotides, and so on, going up to thefull length nucleic acid. Similarly, a portion of a polypeptide may be 4amino acids, 5 amino acids, 6 amino acids, 7 amino acids, and so on,going up to the full length polypeptide. The length of the portion to beused will depend on the particular application. A portion of a nucleicacid useful as hybridization probe may be as short as 12 nucleotides; inone embodiment, it is 20 nucleotides. A portion of a polypeptide usefulas an epitope may be as short as 4 amino acids. A portion of apolypeptide that performs the function of the full-length polypeptidewould generally be longer than 4 amino acids.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences includes reference to the residuesin the two sequences which are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences which differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well-known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., according to the algorithm of Meyersand Miller, Computer Applic. Biol. Sci., 4:11-17 (1988).

As used herein, the term “substantially complementary” means that twonucleic acid sequences have at least about 65%, preferably about 70%,more preferably about 80%, even more preferably 90%, and even morepreferably about 98%, about 98.5%, about 99%, about 99.1%, about 99.2%,about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about99.8%, or about 99.9%, sequence complementarity to each other. Thismeans that primers and probes must exhibit sufficient complementarity totheir template and target nucleic acid, respectively, to hybridize understringent conditions. Therefore, the primer and probe sequences need notreflect the exact complementary sequence of the binding region on thetemplate and degenerate primers can be used. For example, anon-complementary nucleotide fragment may be attached to the 5′-end ofthe primer, with the remainder of the primer sequence beingcomplementary to the strand. Alternatively, non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer has sufficient complementarity with the sequence of one of thestrands to be amplified to hybridize therewith, and to thereby form aduplex structure which can be extended by the polymerizing means. Thenon-complementary nucleotide sequences of the primers may includerestriction enzyme sites. Appending a restriction enzyme site to theend(s) of the target sequence would be particularly helpful for cloningof the target sequence. A substantially complementary primer sequence isone that has sufficient sequence complementarity to the amplificationtemplate to result in primer binding and second-strand synthesis. Theskilled person is familiar with the requirements of primers to havesufficient sequence complementarity to the amplification template.

The terms “substantially pure” and “isolated,” are used interchangeablyand describe anything which is substantially separated from otherthings. For example, it can be used to refer to a fungus, a part or formof a fungus (e.g., a conidia, pycnidia, chlamydospores, and hyphae), aprotein, a peptide or a nucleic acid which is substantially separatedfrom other cellular and/or (sub)cellular components or contaminantswhich naturally accompany it. The term embraces fungi, a fungal form orpart, a nucleic acid or a protein which has been removed from itsnaturally occurring environment. Generally, the term refers to purifiedthings having a purity of at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 85%, at least about90%, at least about 95%, at least about 98%, or at least about 99% byweight. Minor variants or chemical modifications typically share thesame polypeptide or nucleotide sequence. A substantially pure protein ornucleic acid will typically comprise about 85 to 100% (w/w) of a proteinor nucleic acid sample, more usually about 95%, and preferably will beover about 99% pure. Protein or nucleic acid purity or homogeneity canbe indicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a protein sample, followed byvisualizing a single polypeptide band on a polyacrylamide gel uponstaining, or by agarose gel electrophoresis of a nucleic acid sample,followed by visualizing a single polynucleotide band on an agarose gelupon staining. “Staining” may either refer to the use of α-specificpeptide or nucleic acid stains such as silver and Coomassie stains, orethidium bromide and SYBR® stains, or may refer to the use of specificpeptide or nucleic acid stains such as contacting the peptide with anantibody and visualizing the antibody using a labeled secondary antibody(e.g. conjugated to alkaline phosphatase) in the case of proteins orpeptides, or contacting the nucleic acid with a complementary probelabeled for visualizing the presence of hybridization between thenucleic acid and the probe. For certain purposes higher resolution canbe provided by using high performance liquid chromatography (HPLC) or asimilar means for purification. Such methods are in the area of commongeneral knowledge (see e.g. Katz, et al., 1998).

The terms “stringency” or “stringent hybridization conditions” refer tohybridization conditions that affect the stability of hybrids, e.g.,temperature, salt concentration, pH, formamide concentration and thelike. These conditions are empirically optimized to maximize specificbinding and minimize non-specific binding of primer or probe to itstarget nucleic acid sequence. The terms as used include reference toconditions under which a probe or primer will hybridize to its targetsequence, to a detectably greater degree than other sequences (e.g. atleast 2-fold over background). Stringent conditions are sequencedependent and will be different in different circumstances. Longersequences hybridize specifically at higher temperatures. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The T m is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe or primer. Typically, stringentconditions will be those in which the salt concentration is less thanabout 1.0 M Na+ion, typically about 0.01 to 1.0 M Na+ ion concentration(or other salts) at pH 7.0 to 8.3 and the temperature is at least about30° C. for short probes or primers (e.g. 10 to 50 nucleotides) and atleast about 60° C. for long probes or primers (e.g. greater than 50nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringent conditions or “conditions of reduced stringency” includehybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDSat 37° C. and a wash in 2×SSC at 40° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60° C. Hybridization procedures arewell known in the art and are described by e.g. Ausubel et al., 1998 andSambrook et al., 2001.

As used herein, “coding sequence” refers to a DNA sequence that codesfor a specific amino acid sequence. “Regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence.

As used herein, “regulatory sequences” may include, but are not limitedto, promoters, translation leader sequences, introns, andpolyadenylation recognition sequences.

As used herein, “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Thepromoter sequence consists of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence that can stimulate promoteractivity, and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Promoters may be derived in their entirety from a native gene,or be composed of different elements derived from different promotersfound in nature, or even comprise synthetic DNA segments. It isunderstood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, DNA fragments of some variation may have identicalpromoter activity. Promoters that cause a gene to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”.

As used herein, the “3′ non-coding sequences” refer to DNA sequenceslocated downstream of a coding sequence and include polyadenylationrecognition sequences and other sequences encoding regulatory signalscapable of affecting mRNA processing or gene expression. Thepolyadenylation signal is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor. The use of different 3′ non-coding sequences is exemplifiedby Ingelbrecht, I. L., et al. (1989) Plant Cell 1:671-680.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is regulated by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of regulatingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of theinvention can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

As used herein, the term “recombinant” refers to an artificialcombination of two otherwise separated segments of sequence, e.g., bychemical synthesis or by the manipulation of isolated segments ofnucleic acids by genetic engineering techniques.

As used herein, the phrases “recombinant construct”, “expressionconstruct”, “chimeric construct”, “construct”, and “recombinant DNAconstruct” are used interchangeably herein. A recombinant constructcomprises an artificial combination of nucleic acid fragments, e.g.,regulatory and coding sequences that are not found together in nature.For example, a chimeric construct may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. Such constructmay be used by itself or may be used in conjunction with a vector. If avector is used then the choice of vector is dependent upon the methodthat will be used to transform host cells as is well known to thoseskilled in the art. For example, a plasmid vector can be used. Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells comprising any of the isolated nucleic acidfragments of the invention. The skilled artisan will also recognize thatdifferent independent transformation events will result in differentlevels and patterns of expression (Jones et al., (1985) EMBO J.4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, or phenotypicanalysis, among others. The term “expression”, as used herein, refers tothe production of a functional end-product e.g., a mRNA or a protein(precursor or mature).

As used herein, the term “transformation” refers to the transfer ofnucleic acid (i.e., a nucleotide polymer) into a cell. As used herein,the term “genetic transformation” refers to the transfer andincorporation of DNA, especially recombinant DNA, into a cell.

As used herein, the term “transformant” refers to a cell, tissue ororganism that has undergone transformation. The original transformant isdesignated as “T0” or “T₀.” Selfing the T0 produces a first transformedgeneration designated as “T1” or “T₁.”

As used herein, the term “transgene” refers to a nucleic acid that isinserted into an organism, host cell or vector in a manner that ensuresits function.

As used herein, the term “transgenic” refers to cells, cell cultures,organisms (e.g., plants), and progeny which have received a foreign ormodified gene by one of the various methods of transformation, whereinthe foreign or modified gene is from the same or different species thanthe species of the organism receiving the foreign or modified gene.

As used herein, “antisense inhibition” or “antisense silencing” refersto the production of antisense RNA transcripts capable of suppressingthe expression of the target protein. “Co-suppression” refers to theproduction of sense RNA transcripts capable of suppressing theexpression of identical or substantially similar foreign or endogenousgenes (U.S. Pat. No. 5,231,020).

As used herein, the term “vector”, “plasmid”, or “construct” refersbroadly to any plasmid or virus encoding an exogenous nucleic acid. Theterm should also be construed to include non-plasmid and non-viralcompounds which facilitate transfer of nucleic acid into virions orcells, such as, for example, polylysine compounds and the like. Thevector may be a viral vector that is suitable as a delivery vehicle fordelivery of the nucleic acid, or mutant thereof, to a cell, or thevector may be a non-viral vector which is suitable for the same purpose.Examples of viral and non-viral vectors for delivery of DNA to cells andtissues are well known in the art and are described, for example, in Maet al. (1997, Proc. Natl. Acad. Sci. U.S.A. 94:12744-12746). Examples ofviral vectors include, but are not limited to, a recombinant vacciniavirus, a recombinant adenovirus, a recombinant retrovirus, a recombinantadeno-associated virus, a recombinant avian pox virus, and the like(Cranage et al., 1986, EMBO J. 5:3057-3063; International PatentApplication No. WO94/17810, published Aug. 18, 1994; InternationalPatent Application No. WO94/23744, published Oct. 27, 1994). Examples ofnon-viral vectors include, but are not limited to, liposomes, polyaminederivatives of DNA, and the like.

As used herein, the phrase “ligno-cellulosic feedstocks” refers tofeedstocks containing ligno cellulose. Non-limiting examples ofligno-cellulosic feedstocks include, agricultural crop residues (e.g.,wheat straw, barley straw, rice straw, small grain straw, corn stover,corn fibers (e.g., corn fiber gum (CFG), distillers dried grains (DDG),corn gluten meal (CGM)), switch grass, hay-alfalfa, sugarcane bagasse),non-agricultural biomass (e.g., algal mats, urban tree residue), forestproducts and industry residues (e.g., softwood first/secondary millresidue, hard softwood first/secondary mill residue, recycled paper pulpsludge), ligno-cellulosic containing waste (e.g., newsprint, wastepaper, brewing grains, used rubber tire (UTR), municipal organic waste,yard waste, clinical organic waste, waste generated during theproduction of biofuels (e.g. processed algal biomass, glycerol), and acombination thereof.

As used herein, unless otherwise specified, the term “carbohydrate”refers to a compound of carbon, hydrogen, and oxygen that contains analdehyde or ketone group in combination with at least two hydroxylgroups. The carbohydrates of the present invention can also beoptionally substituted or deoxygenated at one or more positions.Carbohydrates thus include substituted and unsubstitutedmonosaccharides, disaccharides, oligosaccharides, and polysaccharides.The saccharide can be an aldose or ketose, and may comprise 3, 4, 5, 6,or 7 carbons. In one embodiment they are monosaccharides. In anotherembodiment they can be pyranose and furanose sugars. They can beoptionally deoxygenated at any corresponding C-position, and/orsubstituted with one or more moieties such as hydrogen, halo, haloalkyl,carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino,dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid,thiol, imine, sulfonyl, sulfanyl, sulfinyl, sulfamonyl, ester,carboxylic acid, amide, phosphonyl, phosphinyl, phosphoryl, thioester,thioether, oxime, hydrazine, carbamate. These saccharide units can bearranged in any order and the linkage between two saccharide units canoccur in any of approximately ten different ways. As a result, thenumber of different possible stereoisomeric oligosaccharide chains isenormous. In one embodiment, said carbohydrates are selected from thegroup consisting of monosaccharides, disaccharides, oligosaccharides,polysaccharides, and a combination thereof.

As used herein, the term “monosaccharide” refers to sugar monomersselected from the group consisting of three-carbon sugars (trioses),four-carbon sugars (tetroses), five-carbon sugars (pentoses), six-carbonsugars (hexoses), et al., and a combination thereof. In one embodiment,the five-carbon sugars are selected from the group consisting ofketopentose (e.g., ribulose, xylulose), aldopentose (ribose, arabinose,xylose, lyxose), deoxy sugar (deoxyribose), and a combination thereof.In one embodiment, the six-carbon sugars are selected from the groupconsisting of aldohexoses (e.g., allose, altrose, glucose, mannose,glucose, idose, galactose, talose), cyclic hemiacetals, ketohexoses(e.g., psicose, fructose, sorbose, tagatose). In one embodiment, saidmonosaccharides are selected from the group consisting of trioses,tetroses, pentoses, hexoses, heptoses, et al., and a combinationthereof. In one embodiment, the monosaccharides are in linear form; inanother embodiment, the monosaccharides are in cyclic form.

As used herein, the phrase “fermentable sugars” refers to sugarcompounds that can be converted to useful value-added fermentationproducts, non-limiting examples of which include amino acids, vitamins,pharmaceuticals, animal feed supplements, specialty chemicals, chemicalfeedstocks, plastics, solvents, fuels, or other organic polymers, lacticacid, and ethanol, including fuel ethanol. Specific value-added productsthat may be produced by the methods of the invention include, but notlimited to, biofuel (including ethanol, butanol and isobutanol); lacticacid; plastics; specialty chemicals; organic acids, including citricacid, succinic acid and maleic acid; solvents; animal feed supplements;pharmaceuticals; vitamins; amino acids, such as lysine, methionine,tryptophan, threonine, and aspartic acid; industrial enzymes, such asproteases, cellulases, amylases, glucanases, lactases, lipases, lyases,oxidoreductases, transferases and xylanases; and chemical feedstocks.

As used herein, the term “fungus” or “fungi” refers to a distinct groupof eukaryotic, spore-forming organisms with absorptive nutrition andlacking chlorophyll.

As used herein, the term “acidification material” refers to anymaterials, chemical compounds, agents, compositions which when addedinto a solvent (e.g., water), gives a solution with a hydrogen ionactivity greater than in pure solvent (e.g., water). The material can bein gas, liquid, or solid form. The material can be organic and/orinorganic. Non-limiting examples of acidification material include anymaterial comprises hydrogen halides and their solutions (e.g.,hydrochloric acid (HCl), hydrobromic acid (HBr), hydroiodic acid (HI)),halogen oxoacids (e.g., hypochloric acid, chloric acid, perchloric acid,periodic acid and corresponding compounds for bromine and iodine),sulfuric acid (H₂SO₄), fluorosulfuric acid, nitric acid (HNO₃),phosphoric acid (H₃PO₄), fluoroantimonic acid, fluoroboric acid,hexafluorophosphoric acid, chromic acid (H₂CrO₄), sulfonic acids,methanesulfonic acid (aka mesylic acid, MeSO₃H), ethanesulfonic acid(aka esylic acid, EtSO₃H), benzenesulfonic acid (aka besylic acid,PhSO₃H), p-toluenesulfonic acid (aka tosylic acid) (CH₃C₆H₄SO₃H),trifluoromethanesulfonic acid (aka triflic acid, CF₃SO₃H), carboxylicacids (e.g., acetic acid, citric acid, formic acid, gluconic acid,lactic acid, oxalic acid, tartaric acid), Vinylogous carboxylic acids(e.g., ascorbic acid, meldrum's acid), acid salts (e.g., sodiumbicarbonate (NaHCO₃), sodium hydrosulfide (NaHS), sodium bisulfate(NaHSO₄), monosodium phosphate (NaH₂PO₄), and disodium phosphate(Na₂HPO₄)).

As used herein, the term “energy-rich metabolites” refers to metabolitesproduced by an organism (e.g., a fungus), wherein the metabolite can bedirectly used as biofuel (e.g., alcohol), or as precursor for biofuelproduction (e.g., lipids for biodiesel).

As used herein, the term “neutralize”, “neutralizing”, and“neutralization” refers to a chemical reaction in aqueous solutions,wherein an acid and a base react to form water and a salt, and whereinthe pH of the solution is brought closer to 7.

As used herein, the term “manganese donor” refers to a composition orcompound which can provide manganese ion (e.g., manganese(I),manganese(II), and manganese(III)) in an aqueous solution. Non-limitingexample of manganese donors include, Mn₂(CO)₁₀, K₅[Mn(CN)₆NO, MnCl₂,MnF₂, MnBr₂, MnO, MnO₂, MnCl₃, MnF₃, MnBr₃, MnCO₃, Mn(CH₃COO)₂,C₆H₉MnO₆, MnTiO₃, [CH₃COCH═C(O)CH₃]₂Mn, [C₆H₁₁(CH₂)₃CO₂]₂Mn, (HCO₂)₂Mn,Mn(C₅HF₆O₂)₂, Mn(PH₂O₂)₂, MnI₂, (C₃H₅O₃)₂Mn, MnMoO₄, Mn(NO₃)₂,Mn(ClO₄)₂, C₃₂H₁₆MnN₈, MnSO₄, (CH₃COO)₃Mn, C₃₂H₁₆ClMnN₈, C₄₈H₂₈ClMnN₄O₈,C₅H₄CH₃Mn(CO)₃, Mn(C₅H₄C₂H₅)₂, and C₁₆H₂₂Mn.

As used herein, the phrase “pH buffering materials” refers to refers tocompositions that when added in a liquid mixture, can maintain the pH ofsaid liquid mixture wherein the pH is kept around about 0.5, about 0.6,about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9,about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2,about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5,about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8,about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about6.5 about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0. Forexample, the pH of the liquid mixture is in a range between about 0.5 toabout 3.0. Such composition can comprise compounds such as acid, acidsalts, basic and basic salts, for example, HCl, H₂NO₃, H₂SO₄, NaHCO₃,NaHS, NaHSO₄, NaH₂PO₄, Na₂HPO₄, NaHSO₃, KHCO₃, KHS, KHSO₄, KH₂PO₄,K₂HPO₄, KHSO₃, NaOH, KOH, Mg(OH)₂, Na₂CO₃, K₂CO₃, KHCO₃, CaCO₃, MgCO₃,Na₂S, K₂S et al.

As used herein, the term “aerobic conditions” refers to conditions wheresufficient oxygen, is provided, and anaerobic respiration in amicroorganism growing under such conditions is prohibited.

As used herein, the term “substantially aerobic conditions” refers toconditions wherein the supply of oxygen is limited, but the cellularrespiration in an organism is dominantly aerobic respiration.

As used herein, the term “biofuel” (also called bioenergy) is defined assolid, liquid or gaseous fuel derived from relatively recently dead ordying biological material and is distinguished from fossil fuels, whichare derived from long dead biological material. It can be produced fromany biological carbon source theoretically. Biofuels can be classifiedinto first generation biofuels (which are made from sugar, starch,vegetable oil, and animal fats, including but not limited to vegetableoil, biodiesel, bioalcohols, bioethers, biogas, syngas and solidbiofuels), second generation biofuels (which are produced from biomassof non food crops, also called cellulosic biofuels, including but notlimited to, biohydrogen, biomethanol, DMF, Bio-DME, Fischer-Tropschdiesel, biohydrogen diesel, mixed alcohols and wood diesel), and thirdgeneration biofuels (also called algae fuels, which are made fromalgae).

The term “biofuel precursor” refers to an organic molecule in which allcarbon contained within is derived from biomass and is biochemicallyconverted. It can be further converted either chemically orbiochemically, into a biofuel. For example, a biofuel precursorincludes, but is not limited to, e.g. isobutanol, isopropanol, propanol,2-butanol, butanol, pentanol, 2-pentanol, 3-pentanol,3-methyl-1-butanol, 2-methyl-1-butanol, 3-methyl-2-butanol, and lipid.

As used herein, the term “biodiesel” refers to a vegetable oil or animalfat-based diesel fuel consisting of long chain alkyl (e.g., methyl,propyl or ethyl) esters. It can be made by chemically reacting lipidswith one or more types of alcohol in a transesterification reaction.Chemically it comprises a mix of mono-alkyl esters of long chain fattyacids. Alcohols that can be used to produce biodiesel include, but arenot limited to, methanol, ethanol, propanol, isopropanol, butanol,isobutanol, and 2-ethoxyethanol. Acidic or alkaline catalyst can beapplied to facilitate esterification of fatty acids. Glycerol isproduced as a by-product in such reactions.

As used herein, the phrase “fatty acids” refers to long-chainedmolecules having a methyl group at one end and a carboxylic acid groupat the other end.

As used herein, the phrase “isolated fungus” refers to any compositioncomprising a fungus population which is obtained from a natural source.

As used herein, the term “bioalcohols” refers to alcohols synthesizedbiologically, including, but not are not limited to, bioethanol,biomethanol, biobutanol, and other bioalcohols.

As used herein, the term “alcohol” refers to any organic compound inwhich a hydroxyl group (—OH) is bound to a carbon atom of an alkyl orsubstituted alkyl group. An important group of alcohols is formed by thesimple acyclic alcohols, the general formula for which isC_(n)H_(2n+1)OH.

As used herein, the term “biogas” refers to a gas produced by thebiological breakdown of organic matter in the absence of oxygen. Biogasoriginates from biogenic material and is a type of biofuel.

As used herein, the term “siderophores” refers to small, high-affinitymetal ion chelating compounds secreted by microorganisms such asbacteria, fungi and grasses.

As used herein, the term “feedstock” refers to a raw material or mixtureof raw materials supplied to a biocatalyst or fermentation process fromwhich other products can be made. For example, a carbon source, such asbiomass or the carbon compounds derived from biomass, is a feedstock fora biocatalyst that produces a biofuel and/or biofuel precursor in afermentation process. In addition, a feedstock may contain nutrientsother than a carbon source. Feedstock can also be municipal, industrial,and farm sewage waste,

As used herein, the term “biocatalyst” refers to a living system or cellof any type that speeds up chemical reactions by lowering the activationenergy of the reaction and is neither consumed nor altered in theprocess. Biocatalysts may include, but are not limited to,microorganisms such as yeasts, fungi, bacteria, and archaea. Forexample, the isolated fungal strain of the present invention can be usedas a biocatalyst in the production of biofuels.

As used herein, the term “fermentation” or “fermentation process” refersa process in which a biocatalyst is cultivated in a culture mediumcontaining raw materials, such as feedstock and nutrients, wherein thebiocatalyst converts raw materials, such as a feedstock, into products.

As used herein, the term “carbon source” generally refers to a substancesuitable to be used as a source of carbon for prokaryotic or eukaryoticcell growth. Carbon sources include, but are not limited to, biomasshydrolysates, carbohydrates (e.g., starch, sucrose, polysaccharides, andmonosaccharides), cellulose, hemicellulose, xylose, and lignin, as wellas monomeric components of these substrates. Carbon sources can comprisevarious organic compounds in various forms, including, but not limitedto polymers, carbohydrates, acids, alcohols, aldehydes, ketones, aminoacids, peptides, etc. These include, for example, variousmonosaccharides such as glucose, dextrose (D-glucose), maltose,oligosaccharides, polysaccharides, saturated or unsaturated fatty acids,succinate, lactate, acetate, ethanol, etc., or mixtures thereof.Photosynthetic organisms can additionally produce a carbon source as aproduct of photosynthesis.

As used herein, the term “biomass” refers to biological material derivedfrom living, or recently living organisms, e.g., stems, leaves, andstarch-containing portions of green plants, or wood, waste, forestresidues (dead trees, branches and tree stumps), yard clippings, woodchips, or materials derived from algae or animals, and is mainlycomprised of starch, lignin, pectin, cellulose, hemicellulose, and/orpectin. Biomass may also include biodegradable wastes that can be burntas fuel. It excludes organic material such as fossil fuel which has beentransformed by geological processes into substances such as coal orpetroleum. Biomass can be decomposed by either chemical or enzymatictreatment to the monomeric sugars and phenols of which it is composed(Wyman, C. E. 2003 Biotechnological Progress 19:254-62). This resultingmaterial, called biomass hydrolysate, is neutralized and treated toremove trace amounts of organic material that may adversely affect thebiocatalyst, and is then used as a feedstock for fermentations using abiocatalyst.

As used herein, the term “cellulosic biomass” refers to biomass composedprimarily of plant fibers that are inedible or nearly inedible by humansand have cellulose as a prominent component. There fibers may behydrolyzed to yield a variety of sugars that can be fermented bymicroorganisms. Examples of cellulosic biomass include grass, wood, andcellulose-rich residues resulting from agriculture or the forestproducts industry.

As used herein, the term “starch” refers to a polymer of glucose readilyhydrolyzed by digestive enzymes. Starch is usually concentrated inspecialized portions of plants, such as potatoes, corn kernels, ricegrains, wheat grains, and sugar cane stems.

As used herein, the term “lignin” refers to a polymer material, mainlycomposed of linked phenolic monomeric compounds, such as p-coumarylalcohol, coniferyl alcohol, and sinapyl alcohol, which forms the basisof structural rigidity in plants and is frequently referred to as thewoody portion of plants. Lignin is also considered to be thenon-carbohydrate portion of the cell wall of plants.

As used herein, the term “cellulose” refers to a long-chain polymerpolysaccharide carbohydrate of beta-glucose of formula (C₆H₁₀O₅)_(n),usually found in plant cell walls in combination with lignin and anyhemicellulose.

As used herein, the term “hemicellulose” refers to a class of plantcell-wall polysaccharides that can be any of several heteropolymers.These include xylan, xyloglucan, arabinoxylan, arabinogalactan,glucuronoxylan, glucomannan and galactomannan. Monomeric components ofhemicellulose include, but are not limited to: D-galactose, L-galactose,D-mannose, L-rhamnose, L-fucose, D-xylose, L-arabinose, and D-glucuronicacid. This class of polysaccharides is found in almost all cell wallsalong with cellulose. Hemicellulose is lower in weight than celluloseand cannot be extracted by hot water or chelating agents, but can beextracted by aqueous alkali. Polymeric chains of hemicellulose bindpectin and cellulose in a network of cross-linked fibers forming thecell walls of most plant cells.

The term “pectin” as used herein refers to a class of plant cell-wallheterogeneous polysaccharides that can be extracted by treatment withacids and chelating agents. Typically, 70-80% of pectin is found as alinear chain of α-(1-4)-linked D-galacturonic acid monomers. The smallerRG-I fraction of pectin is comprised of alternating (1-4)-linkedgalacturonic acid and (1-2)-linked L-rhamnose, with substantialarabinogalactan branching emanating from the rhamnose residue. Othermonosaccharides, such as D-fucose, D-xylose, apiose, aceric acid, Kdo,Dha, 2-O-methyl-D-fucose, and 2-O-methyl-D-xylose, are found either inthe RG-II pectin fraction (<2%), or as minor constituents in the RG-Ifraction. Proportions of each of the monosaccharides in relation toD-galacturonic acid vary depending on the individual plant and itsmicro-environment, the species, and time during the growth cycle. Forthe same reasons, the homogalacturonan and RG-I fractions can differwidely in their content of methyl esters on GalA residues, and thecontent of acetyl residue esters on the C-2 and C-3 positions of GalAand neutral sugars.

As used herein, the phrase “facultative anaerobic organism” or a“facultative anaerobic microorganism” or a “facultative anaerobicbiocatalyst” is defined as an organism that can grow in either thepresence or in the absence of oxygen, such as the fungal strainsisolated in the present invention.

As used herein, the term “byproduct” means an undesired product relatedto the production of biofuel and/or biofuel precursor. Byproducts aregenerally disposed as waste, adding cost to a process.

As used herein, the term “co-product” means a secondary or incidentalproduct related to the production of biofuel and/or biofuel precursor.Co-products have potential commercial value that increases the overallvalue of biofuel precursor production, and may be the deciding factor asto the viability of a particular biofuel precursor production process.

As used herein, the term “distillers dried grains”, abbreviated as DDG,refers to the solids remaining after a fermentation, usually consistingof unconsumed feedstock solids, remaining nutrients, protein, fiber, andoil, as well as biocatalyst cell debris. The term may also includesoluble residual material from the fermentation and is then referred toas “distillers dried grains and solubles” (DDGS). DDG or DDGS are anexample of a co-product from a biofuel precursor production process.

As used herein, the term “nutrient” is defined as a chemical compoundthat is used by a biocatalyst to grow and survive. Nutrients can beorganic compounds such as carbohydrates and amino acids or inorganiccompound such as metal salts.

As used herein, the term “complex nutrient” is defined as a nutrientsource containing mostly monomeric organic compounds used by abiocatalyst for the production of proteins, DNA, lipids, andcarbohydrates. The term “rich nutrient” is used interchangeablythroughout with the term complex nutrient. Typically, complex nutrientsor rich nutrients are derived from biological materials, such asslaughterhouse waste, dairy wastes, or agricultural residues. Complexnutrients or rich nutrients include, but are not limited to: yeastextract, tryptone, peptone, soy extract, corn steep liquor, soy protein,and casein.

As used herein, the phrase “aerobic metabolism” refers to a biochemicalprocess in which oxygen is used to make energy, typically in the form ofATP, from carbohydrates. Typical aerobic metabolism occurs viaglycolysis and the TCA cycle, wherein a single glucose molecule ismetabolized completely into carbon dioxide in the presence of oxygen.

As used herein, the phrase “anaerobic metabolism” refers to abiochemical process in which oxygen is not the final acceptor ofelectrons contained in NADH. Anaerobic metabolism can be divided intoanaerobic respiration, in which compounds other than oxygen serve as theterminal electron acceptor, and fermentation, in which the electronsfrom NADH are utilized to generate a reduced product via a “fermentativepathway.”

As used herein, the term “recombinant microorganism” and “recombinanthost cell” are used interchangeably and refer to microorganisms thathave been genetically modified to express or over-express endogenouspolynucleotides, or to express heterologous polynucleotides, such asthose included in a vector, or which have a reduction in expression ofan endogenous gene. The polynucleotide generally encodes a target enzymeinvolved in a metabolic pathway for producing a desired metabolite. Itis understood that the terms “recombinant microorganism” and“recombinant host cell” refer not only to the particular recombinantmicroorganism but to the progeny or potential progeny of such amicroorganism. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

As used herein, the term “fermentation” refers to a method to producebiofuel and other products where biomass (pretreated or unpretreated)was fermented by microorganisms (e.g., bacteria, cyanobacteria, yeast,fungi or algae).

As used herein, the terms “microbiological fermentation” refers to aprocess where organic substances are broken down and re-assembled intoproducts by microorganisms. The substances may include, but not limitedto, glucose, sucrose, glycerol, starch, maltodextrine, lactose, fats,hydrocarbons, protein, ammonia, nitrate, phosphorus sources. Theproducts may include, but not limited to, traditional products(including but not limited to, bread, beer, wine, spirits, cheese, dairyproducts, fermented meats and vegetables, mushrooms, soy sauce andvinegar), agricultural products (including but not limited to,gibberellins, fungicides, insecticides, silage, amino acids such asL-Glutamine, L-Lysine, L-Tryptophan, L-Throenine, L-aspartic (+),L-arylglycines), enzymes (including but not limited to, carbohydrates,celluloses, lipases, pectinases, proteases), fuels and chemicalfeedstocks (including but not limited to, acetone, butanol, butanediol,isopropanol, ethyl alcohol, glycerol, methane, glycerol, butyric acid,methane, citric acid, fumaric acid, lactic acid, propionic acid,succinic acid, itaconic acid, acetic acid, 3-hydroxypropionic acid,glyconic acid, tartaric acid and L-glutaric acid or salts of any ofthese acids), nucleotides, organic acids, pharmaceuticals and relatedcompounds (including but not limited to, alkaloids, antibiotics,hormones, immunosuppressant, interferon, steroids, vaccines, vitamins)and polymers (including but not limited to, alginates, dextran, gellan,polyhydroxybutyrate, scleroglucan and xanthan). The microorganisms usedfor fermentation may include both prokaryotic microorganisms (includingbacteria, cyanobacteria) and eukaryotic microorganisms (including yeast,fungi and algae).

As used herein, the phrase “energy crops” refers to plants grown as alow cost and low maintenance harvest used to make biofuels, or directlyexploited for its energy content. Commercial energy crops are typicallydensely planted, high yielding crop species where the energy crops willbe burnt to generate power. Woody crops such as Willow or Poplar arewidely utilized as well as tropical grasses such as Miscanthus andPennisetum purpureum (both known as elephant grass). If carbohydratecontent is desired for the production of biogas, whole-crops such asmaize, Sudan grass, millet, white sweet clover and many others, can bemade into silage and then converted into biogas.

As used herein, the term micro-aerobic refers to an environment in whichthe concentration of oxygen is less than that in air, but cellularrespiration in an organism is dominantly aerobic respiration.

Fusarium

Fusarium species may produce three types of spores called macroconidia,microconidia, and chlamydospores. The macrocodinia are produced in aspecialized structure called a sporodochium in which the spore mass issupported by a superficial cushionlike mass of short monophialidesbearing the macroconidia, or produced on monophialides and polyphialidesin the aerial mycelium. A monophialide is a condiophore with only oneopening or pore through which endoconidia are extruded, while apolyphialide has two or more such openings or pores. Some conidia areintermediate in size and shape, and these have been referred to as bothmacroconidia and mesoconidia. Microconidia are produced in the aerialmycelium but not in sporodochia. They may be produced in false headsonly or in false heads and chains on either monophialides orpolyphialides. False heads occur when a drop of moisture forms on thetip of the conidiophore and contains the endoconidia as they areproduced. Microconidia are of various shapes and sizes, and thoseproduced in chains have a truncate base. The third type of spore formedby Fusarium species is a chlamydospore, which is a thick-walled sporefilled with lipid-like material that serves to carry the fungus overwinter in soil when a suitable host is not available. The chlamydosporesmay be borne singly, in pairs, in clumps, or in chains, and the outerwall may be smooth or rough. Morphology of these spores can be used toseparate species in Fusarium.

Methods of transferring and culturing Fusarium species are well known inthe art. For example, the single-spore method and the hyphal-tip methodare the primary means used to initiate and transfer cultures of Fusariumspecies. The hyphal-tip method is used to transfer cultures of Fusariumspecies that mutate rapidly after being transferred by single conidia.The transfer is done under the dissecting microscope, in much the samemanner as the transfer of a single germinating conidium. The four mediaused for growing Fusarium species for identification are carnation leafagar (Fisher et al., 1982, Carnation leaves as a substrate and forpreserving Fusarium species. Phytopathology 72:151-153), potato dextroseagar (Nelson et al., 1983. Fusarium species: an illustrated manual foridentification. Pennsylvania State University Press, University Park),KCl medium (Fisher et al., 1983. Taxonomic importance of microconidialchains in Fusarium section Liseola and effects of water potential ontheir formation. Mycologia 75:693-698), and soil agar (Klotz et al.,1988. A medium for enhancement of chlamydospore formation in Fusariumspecies. Mycologia 80:108-109), each of which is incorporated byreference in its entirety.

The majority of Fusarium species isolated from nature produce theirmacroconidia on sporodochia. The sporodochial type often mutates inculture, especially on media rich in carbohydrates. Mutations may alsooccur in nature, but are rare. These mutants may give rise to others sothat a mutational sequence occurs. In pathogenic isolates, these mutantsfrequently exhibit a loss of virulence and the ability to produce toxinsmay be reduced or lost. The two major types of mutants produced from thesporodochial type are the pionnotal type (e.g., the production of littleor no aerial mycelium; production of abundant macroconidia on thesurface of the colony causing the surface to appear shiny and wet; moreintense pigmentation of colonies than of the sporodochial colonies; theproduction of longer and thinner macroconidia than those produced by thesporodochial type; less virulent than the sporodochial type and may alsolose the ability to produce toxins) and the mycelial type (e.g., theproduction of abundant aerial mycelium; the production of very few to nomacroconidia; the frequent lack of sporodochia, sclerotia andpigmentation in culture; mutants that may be less virulent than thesporodochial type and may also lose the ability to produce toxins).

Procedures that reduce mutant populations include, but are not limitedto, initiating cultures from single conidia; initiating cultures fromsingle hyphal tips; avoiding media rich in carbohydrates; and keepingsubculturing to a minimum, as discussed in Nelson et al. (1983, Fusariumspecies: an illustrated manual for identification. Pennsylvania StateUniversity Press, University Park).

Non-limiting example of Fusarium species include, Fusarium angustum(synonym of F. oxysporum), Fusarium aquaeductuum (e.g., Fusariumaquaeductuum var. dimerum, synonym of F. dimerum), Fusarium aquaeductuumvar. media (e.g., Fusarium bostrycoides, synonym of F. oxysporum,Fusarium chlamydosporum (synonym of Dactylium fusarioides, F.fusarioides, F. sporotrichioides var. chlamydosporum, F. tricinctum),Fusarium coeruleum (synonym F. solani var. coeruleum), Fusariumconglutinans (synonym of F. oxysporum), Fusarium dianthi (synonym of F.oxysporum), Fusarium dimerum (synonyms F. aquaeductuum var. dimerum, F.episphaeria), Fusarium episphaeria (synonym of F. dimerum), Fusariumeumartii (synonym of F. solani), Fusarium fujikuroi (synonym of F.moniliforme), Fusarium fusarioides (synonym of F. chlamydosporum),Fusarium illudens (synonym of F. solani), Fusarium incarnatum (synonymof F. semitectum), Fusarium javanicum (synonym of F. solani), Fusariumlini (synonym of F. oxysporum), Fusarium moniliforme (synonym of F.proliferatum, F. verticillioides, F. fujikuroi, F. verticillioides),Fusarium moniliforme var. intermedium (synonym of F. proliferatum),Fusarium napiforme, Fusarium orthoceras (synonym of F. oxysporum),Fusarium oxysporum (synonym of F. angustum, F. bostrycoide,s F.bulbigenum, F. conglutinans, F. dianthi, F. lini, F. orthoceras, F.tracheiphilum, F. vasinfectum), Fusarium pallidoroseum (synonym of F.semitectum), Fusarium proliferatum (synonym of F. moniliforme, F.moniliforme var. intermedium), Fusarium roseum (synonym of F.semitectum), Fusarium roseum var. arthrosporioide (synonym of F.semitectum), Fusarium sacchari, Fusarium semitectum (synonym of F.incarnatum, F. pallidoroseum, F. roseum, F. roseum var. arthrosporioide,Pseudofusarium semitectum), Fusarium solani (synonym of F. eumartii, F.illudens, F. javanicum, F. tumidum, F. ventricosum, Fusisporium solani,Nectria haematococca), Fusarium solani var. coeruleum (synonym of F.coeruleum), Fusarium sporotrichiella var. sporotrichioides (synonym ofF. sporotrichoides), Fusarium sporotrichioides var. chlamydosporum(synonym of F. chlamydosporum), Fusarium sporotrichoides (synonym of F.sporotrichiella var. sporotrichioides, F. tricinctum, Sporotrichellarosea), Fusarium sub glutinous, Fusarium tabacinum (teleomorph ofPlectosphaerella cucumerina), Fusarium tracheiphilum (synonym of F.oxysporum), Fusarium tricinctum (synonym of F. chlamydosporum, F.sporotrichoides), Fusarium tumidum (synonym of F. solani), Fusariumvasinfectum (synonym of F. oxysporum), Fusarium ventricosum (synonym ofF. solani), and Fusarium verticillioides (synonym of F. moniliforme).

More information regarding Fusarium species, methods of identifying,isolating and culturing is described in Nelson et al., (Taxonomy,Biology, and Clinical Aspects of Fusarium Species, 1994, ClinicalMicrobiology Reviews, 7(4): 479-504), Toussoun and Nelson (1976,Fusarium), Booth (Fusarium: laboratory guide to the identification ofthe major species, 1977, Commonwealth Mycological Institute, ISBN0851983839, 9780851983837) and Leslie et al. (The Fusarium laboratorymanual, 2006, Wiley-Blackwell, ISBN 0813819199, 9780813819198), each ofwhich is herein incorporated by reference in its entirety.

Fusarium oxysporum

Fusarium oxysporum, also referred to as pathogen of Panama disease orAgent Green, is a fungus that causes Fusarium wilt disease in more thana hundred species of plants. It does so by colonizing thewater-conducting vessels (xylem) of the plant. As a result of thisblockage and breakdown of xylem, symptoms appear in plants such as leafwilting, yellowing and eventually plant death. Interest in Fusariumoxysporum as a herbicide was first raised after the discovery in the1960s that it was the causative agent in the destruction of the Hawaiiancoca population.

Members of the Fusarium oxysporum species complex (FOSC) are the mostcommon phytopathogenic Fusaria. They cause vascular wilts of over 100cultivated plant species, including tomato, potato, sugarcane, bean,cowpea, date and oil palm, as well as cooking and dessert bananas(Beckman, 1987. The Nature of Wilt Diseases of Plants. The AmericanPhytopathological Society Press, St. Paul). Because it is a long-lived,soil-borne pathogen, infested soil remains contaminated indefinitely, soonly resistant varieties can be grown on that site.

Fusarium oxysporum f sp. lycopersici strain 4287 (race 2, VCG 0030) wasfully sequenced. The genome size is estimated to be 59.9 Mb. Thischaracteristic permits extensive investigation of host-pathogeninteractions, pathogen infection, and plant defense mechanisms, usingthe model plant system A. thaliana. Based on intensive study over thepast 10 years, this strain has displayed remarkable phenotypicstability, including mycelial growth on different culture media,sporulation and high virulence. This strain is also highly amenable totransformation, via both protoplasts and Agrobacterium tumefaciens, andtransformation-mediated gene disruption. A mitotic linkage map wascreated from the protoplast fusion of two strains of F. oxysporum f sp.lycopersici, VCG 0030 (Teunissen et al. 2003, A Near-Isogenic Fusariumoxysporum f sp. lycopersici Strain with a Novel Combination ofAvirulence Characteristics, Phytopathology, 93(11): 1360-1367). A noveland highly efficient gene knock-out technique and molecular cytologicaltools also have been developed (Khang et al., 2005, A dual selectionbased, targeted gene replacement tool for Magnaporthe grisea andFusarium oxysporum. Fungal Genet. Biol. 42:483-492). Microarray analysisfor Fusarium oxysporum is also available (Mcfadden et al., 2006,Fusarium wilt (Fusarium oxysporum f sp. vasinfectum) genes expressedduring infection of cotton (Gossypium hirsutum), Molecular PlantPathology 7(2): 87-101).

Macroscopic morphology of Fusarium oxysporum, may vary significantly ondifferent media. The description below is based upon growth on potatoflakes agar at 25° C. with on/off fluorescent light cycles ofapproximately 12 hours each. Hyphae are septate and hyaline.Conidiophores are short (when contrasted with those of F. solani) andsimple (usually not branched). Macroconidia usually produced abundantly,slightly sickle-shaped, thin-walled, with an attenuated apical cell anda foot-shaped basal cell. They are three to 5-septate measuring23-54×3-4.5 μm. Microconidia are abundant, mostly non-septate,ellipsoidal to cylindrical, slightly curved or straight, 5-12×2.3-3.5 μmoccurring in false heads (a collection of conidia at the tip of thephialide) from short monophialides. Chlamydoconidia are present andoften abundant, occurring singly and in pairs. While F. solani is themost common clinical isolate, Fusarium oxysporum appears to be thesecond most common species recovered. It has been reported in skin andnail infections, in subcutaneous disease, in a neutropenic child managedwith granulocyte colony-stimulating factor, in a disseminated infectionin hemophagocytic lymphohistiocytosis, and in a fatal case involving across reaction with a pan-Candida genus probe. The species is usuallyeasily identified by its lavender color on potato dextrose agar, itsshort monophialides, and microconidia formed only in false heads.

Non-limiting examples of previously isolated Fusarium oxysporum formsinclude, Fusarium oxysporum 247, Fusarium oxysporum CL57, Fusariumoxysporum f cubense, Fusarium oxysporum f perniciosum, Fusariumoxysporum f sp. aechmeae, Fusarium oxysporum f sp. albedinis, Fusariumoxysporum f sp. allii, Fusarium oxysporum f sp. apii, Fusarium oxysporumf sp. arctii, Fusarium oxysporum f sp. asparagi, Fusarium oxysporum fsp. basilici, Fusarium oxysporum f sp. batatas, Fusarium oxysporum f sp.betae, Fusarium oxysporum f sp. bouvardiae, Fusarium oxysporum f sp.bulbigenum, Fusarium oxysporum f sp. callistephi, Fusarium oxysporum fsp. canariensis, Fusarium oxysporum f sp. cannabis, Fusarium oxysporum fsp. carthami, Fusarium oxysporum f sp. cassiae, Fusarium oxysporum f sp.cattleyae, Fusarium oxysporum f sp. cepae, Fusarium oxysporum f sp.chrysanthemi, Fusarium oxysporum f sp. ciceris, Fusarium oxysporum f sp.colocasiae, Fusarium oxysporum f sp. conglutinans, Fusarium oxysporum fsp. cucumerinum, Fusarium oxysporum f sp. cucurbitacearum, Fusariumoxysporum f sp. cyclaminis, Fusarium oxysporum f sp. dianthi, Fusariumoxysporum f sp. elaeidis, Fusarium oxysporum f sp. erythroxyli, Fusariumoxysporum f sp. fabae, Fusarium oxysporum f sp. foetens, Fusariumoxysporum f sp. fragariae, Fusarium oxysporum f sp. gladioli, Fusariumoxysporum f sp. glycines, Fusarium oxysporum f sp. hebes, Fusariumoxysporum f sp. heliotropii, Fusarium oxysporum f sp. koae, Fusariumoxysporum f sp. lactucae, Fusarium oxysporum f sp. lagenariae, Fusariumoxysporum f sp. lentis, Fusarium oxysporum f sp. lilii, Fusariumoxysporum f sp. lini, Fusarium oxysporum f sp. loti, Fusarium oxysporumf sp. luffae, Fusarium oxysporum f sp. lupini, Fusarium oxysporum f sp.lycopersici, Fusarium oxysporum f sp. matthiolae, Fusarium oxysporum fsp. medicaginis, Fusarium oxysporum f sp. melongenae, Fusarium oxysporumf sp. melonis, Fusarium oxysporum f sp. momordicae, Fusarium oxysporum fsp. narcissi, Fusarium oxysporum f sp. nelumbicola, Fusarium oxysporum fsp. niveum, Fusarium oxysporum f sp. opuntiarum, Fusarium oxysporum fsp. passiflorae, Fusarium oxysporum f sp. phaseoli, Fusarium oxysporum fsp. pini, Fusarium oxysporum f sp. pisi, Fusarium oxysporum f sp.radicis-cucumerinum, Fusarium oxysporum f sp. radicis-lycopersici,Fusarium oxysporum f sp. rapae, Fusarium oxysporum f sp. raphani,Fusarium oxysporum f sp. rauvolfiae, Fusarium oxysporum f sp. rhois,Fusarium oxysporum f sp. ricini, Fusarium oxysporum f sp. sesami,Fusarium oxysporum f sp. spinaciae, Fusarium oxysporum f sp. strigae,Fusarium oxysporum f sp. tracheiphilum, Fusarium oxysporum f sp.tulipae, Fusarium oxysporum f sp. vanillae, Fusarium oxysporum f sp.vasinfectum, Fusarium oxysporum f sp. voandzeiae, Fusarium oxysporum fsp. zingiberi, Fusarium oxysporum f tuberosi, Fusarium oxysporum Fo47,Fusarium oxysporum Fo5176, Fusarium oxysporum FOL 4287, Fusariumoxysporum Fov24500, Fusarium oxysporum 115, Fusarium oxysporum MN25,Fusarium oxysporum NRRL 25433, Fusarium oxysporum NRRL 26406, Fusariumoxysporum NRRL 32931, Fusarium oxysporum PHW808, Fusarium oxysporumPHW815, and Fusarium oxysporum var. meniscoideum.

Isolated Fungal Strain

The present invention provides an isolated ligno-cellulose degrading,acidophilic fungal strain of Fusarium oxysporum and its progeny, whereinthe isolated fungal strain and its progeny have at least the followingidentifying characteristics:

-   -   a) the isolated strain is acidophilic and can grow at pH ranging        from about 0.7 to about 7.5; and    -   b) producing lipids from ligno-cellulosic feedstocks, carbon        containing waste products, carbohydrates, or a combination        thereof under aerobic or substantially aerobic conditions.

In one embodiment, the isolated strain of the present invention furthercomprises one or more the following additional identifyingcharacteristics:

-   -   c) producing ethanol and/or hydrogen from ligno-cellulosic        feedstocks, carbon containing waste products, carbohydrates, or        a combination thereof under anaerobic or substantially anaerobic        conditions;    -   d) resisting to Mn concentrations of up to 25 mM, As        concentrations of up to 250 mM or 300 mM, and Hg concentrations        of up to 100 mM;    -   e) being able to dewax wheat straw and other plant material;    -   f) ability to produce lipids from algal feedstocks and from        waste generated during biofuel production (e.g. processed algal        biomass, glycerol) under aerobic or substantially aerobic        conditions;    -   g) ability to produce ethanol and/or hydrogen from algal        feedstocks and from waste generated during biofuel production        (e.g. processed algal biomass, glycerol) under anaerobic or        substantially anaerobic conditions;    -   h) ability to produce Diisooctyl phthalate (DIOP);    -   i) ability to produce Hexanedioic acid, mono(2-ethylhexyl)        ester; and    -   j) comprising an 18S rRNA and ITS region DNA sequence that        shares at least 98% identity to SEQ ID NO. 1.

In one embodiment, said isolated fungal strain of Fusarium oxysporum isthe strain designated as MK7, which has been deposited as ATCC AccessionDeposit No. PTA-10698, or progeny thereof.

In some embodiments, said ligno-cellulosic feedstocks are selected fromthe group consisting of agricultural crop residues (e.g., wheat straw,barley straw, rice straw, small grain straw, corn stover, corn fibers(e.g., corn fiber gum (CFG), distillers dried grains (DDG), corn glutenmeal (CGM)), switch grass, hay-alfalfa, sugarcane bagasse),non-agricultural biomass (e.g., algal mats, urban tree residue), forestproducts and industry residues (e.g., softwood first/secondary millresidue, hard softwood first/secondary mill residue, recycled paper pulpsludge), ligno-cellulosic containing waste (e.g., newsprint, wastepaper, brewing grains, used rubber tire (UTR), municipal organic waste,yard waste, clinical organic waste, waste generated during theproduction of biofuels (e.g. processed algal biomass, glycerol), and acombination thereof. In some embodiments, the carbohydrates are selectedfrom the group consisting of monosaccharides, disaccharides,oligosaccharides, polysaccharides, and mixture thereof.

In some embodiments, said isolated fungal strain and/or its progeny cangrow at a low pH of at most about 7.0, about 6.5, about 6.0, about 5.5,about 5.0, about 4.5, about 4.0, about 3.5, about 2.0, about 1.8, about1.6, about 1.4, about 1.2, about 1.0, about 0.9, about 0.8, or about 0.7or about 0.6, or about 0.5, For example, said fungal strain can grow ata low pH ranging from about 0.7 to about 2.0.

The isolated strain of the present invention can produce lipids at highquantity within the low pH ranges as described above. In one embodiment,the isolated strain can convert feedstock at a higher rate within a lowpH as described above than any other Fusarium oxysporum strains isolatedpreviously in the art. Previously isolated Fusarium oxysporum strainshave been described (see Naim et al., 1985, Bhatia et al., 2006, andNaqvi et al, 1997). In one embodiment, the isolated strain can convertfeedstock to lipids at a rate of at least 0.04 g lipid/g feedstock, 0.05g lipid/g feedstock, 0.06 g lipid/g feedstock, 0.07 g lipid/g feedstock,0.08 g lipid/g feedstock, 0.1 g lipid/g feedstock, 0.12 g lipid/gfeedstock, 0.14 g lipid/g feedstock, 0.16 g lipid/g feedstock, 0.18 glipid/g feedstock, 0.2 g lipid/g feedstock, 0.25 g lipid/g feedstock,0.3 g g lipid/g feedstock, 0.35 g lipid/g feedstock, or 0.4 g lipid/gfeedstock, after 10 days incubation at pH 2.5 under aerobic conditions.

In one embodiment, the isolated fungal strain of the present inventionproduce a more favorable lipid profile compared to previously isolatedfungi or microalgae. For example, the isolated strain produces moresaturated fatty acids (e.g., palitic (16:0) and stearic acids (18:0))and mono-unsaturated fatty acids (e.g., oleic acid (18:1)), but lesspolyunsaturated fatty acids which are more vulnerable to oxidation.

In some embodiments, said isolated fungal strain and/or its progeny cangrow at a high metal concentration, wherein the metal is selected fromthe group consisting Mn, Ag, Zn, Fe, Al, Be, Pb, Cu, Cr, Ni, Cd, Co, Ni,Pd, Pt, U, Th, Mo, Sn, Ti, As, Au, Se, Sb and Hg. In one embodiment, theisolate fungal strain can grow well with a high Mn concentration up to25 mM. In one embodiment, two or more such heavy metal(s) (or heavymetals taken collectively) may be present at more than 0.1 ppm, or 1ppm, or 10 ppm, or 50 ppm, or 100 ppm, or 200 ppm, or 400 ppm, or 500ppm, or 1000 ppm, or 5000 ppm, or 10,000 ppm, or 50,000 ppm, or 100,000ppm, by weight.

The isolated Fusarium oxysporum strain and/or its progeny of the presentinvention can be cultured in the absence of antibiotics with little orno contaminations, wherein said contaminations by other organisms areselected from the group consisting of contaminations by bacteria, otherfungi (e.g., yeasts, molds), algae, plants, insects, and mixturethereon.

The isolated Fusarium oxysporum strain and/or its progeny can be furthermodified, for example, by mutagenesis, and/or recombinant technologies(e.g., transformation). Methods of fungal mutagenesis and recombinanttechnologies are well known in the art. Classical mutagenesis methodscan be applied, which include, but are not limited to, chemicalmutagenesis, insertional mutagenesis, and radiation mutagenesis (e.g.,UV or gamma irradiation). In some cases, genetic crosses can be done tocombine improvements from different mutants.

In one embodiment of the present invention, the isolated fungal strainand/or its progeny is capable of high density cell growth. In someembodiments of the present invention, the microorganisms are capable ofachieving a cell density of at least about 10 g/L, at least about 15g/L, at least about 20 g/L, at least about 25 g/L, at least about 30g/L, at least about 50 g/L, at least about 75 g/L, at least about 100g/L, at least about 125 g/L, at least about 135 g/L, at least about 140g/L, at least about 145 g/L, or at least about 150 g/L. For example, theisolated fungal strain is capable of achieving a cell density of fromabout 10 g/L to about 300 g/L, from about 15 g/L to about 300 g/L, fromabout 20 g/L to about 300 g/L, from about 25 g/L to about 300 g/L, fromabout 30 g/L to about 300 g/L, from about 50 g/L to about 300 g/L, fromabout 75 g/L to about 300 g/L, from about 100 g/L to about 300 g/L, fromabout 125 g/L to about 300 g/L, from about 130 g/L to about 290 g/L,from about 135 g/L to about 280 g/L, from about 140 g/L to about 270g/L, from about 145 g/L to about 260 g/L, from about 150 g/L to about250 g/L, or from about 100 g/L to about 280 g/L. The high density growthof the isolated fungal strain of the present invention can be increasedby adjusting the fermentation conditions (such as temperature, pH,concentration of ions, and gas concentrations).

Genes and Proteins of the Isolated Fungal Strain

Proteins (e.g., certain enzymes) of the wild-type isolated Fusariumoxysporum strain MK7 and/or its progeny can be purified. Methods ofprotein purification are known to one skilled in the art. Detailedprotein purification methods have been described in Janson and Ryden(Protein purification: principles, high-resolution methods, andapplications; Wiley-VCH, 1998, ISBN 0471186260, 9780471186267),Deutscher (Guide to protein purification, Volume 182 of Methods inenzymology, Gulf Professional Publishing, 1990, ISBN 0121820831,9780121820831), and Cutler (Protein purification protocols, Volume 244of Methods in molecular biology, Humana Press, 2004 ISBN 1588290670,9781588290670), which are incorporated by reference in their entiretiesfor all purposes.

Nucleotide sequences of the isolated fungal strain and/or its progeny ofthe present invention can be cloned, sequenced, characterized andmodified through biotechnology. Cloned sequences can be transferred toanother organism.

Non-limiting example of methods of DNA extraction from Fusariumoxysporum and methods of DNA sequence analyses (e.g., PCR, AFLP, SSR andDNA sequence analyses, southern blot, RT-PCT, et al.) have beendemonstrated by Lee et al. (1990, Isolation of DNA from fungal myceliaand single spores, Academic Press, New York, pp. 282-287), Gale et al.(2003, Phytopathology 93:1014-1022), Bogale et al. (2006,Characterization of Fusarium oxysporum isolates from Ethiopia usingAFLP, SSR and DNA sequence analyses, Fungal Diversity, 23:51-65). Inaddition, biological materials, experimental procedures that can be usedfor growing Fusarium oxysporum, RNA isolation, cDNA libraryconstruction, microarray preparation and microarray data analysistechniques have been described by Dowd et al (2004, Gene expressionprofile changes in cotton root and hypocotyl tissues in response toinfection with Fusarium oxysporum f sp. vasinfectum. Mol. Plant-MicrobeInteract. 17:654-667).

General texts which describe molecular biological techniques, which areapplicable to the present invention, such as cloning, mutation, and thelike, include Berger and Kimmel, Guide to Molecular Cloning Techniques,Methods in Enzymology, Vol. 152 Academic Press, Inc., San Diego, Calif.(“Berger”); Sambrook et al., Molecular Cloning—A Laboratory Manual (3rdEd.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2000 (“Sambrook”) and Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (“Ausubel”).These texts describe mutagenesis, the use of vectors, promoters and manyother relevant topics related to, e.g., the cloning and mutating thepresent genes etc. Thus, the invention also encompasses using knownmethods of protein engineering and recombinant DNA technology to improveor alter the characteristics of the proteins expressed in by Fusariumstrains (e.g. strain MK7). Various types of mutagenesis can be used toproduce and/or isolate variant nucleic acids that encode for proteinmolecules and/or to further modify/mutate the proteins of the presentinvention. They include but are not limited to site-directed, randompoint mutagenesis, homologous recombination (DNA shuffling), mutagenesisusing uracil containing templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like. Additional suitable methods include pointmismatch repair, mutagenesis using repair-deficient host strains,restriction-selection and restriction-purification, deletionmutagenesis, mutagenesis by total gene synthesis, double-strand breakrepair, and the like. Mutagenesis, e.g., involving chimeric constructs,is also included in the present invention. In one embodiment,mutagenesis can be guided by known information of the naturallyoccurring molecule or altered or mutated naturally occurring molecule,e.g., sequence, sequence comparisons, physical properties, crystalstructure or the like.

Methods of cloning genes are known in the art. The gene can be cloned asa DNA insert into a vector. The term “vector” refers to the means bywhich a nucleic acid can be propagated and/or transferred betweenorganisms, cells, or cellular components. Vectors include plasmids,viruses, bacteriophages, pro-viruses, phagemids, transposons, artificialchromosomes, and the like, that replicate autonomously or can integrateinto a chromosome of a host cell. A vector can also be a naked RNApolynucleotide, a naked DNA polynucleotide, a polynucleotide composed ofboth DNA and RNA within the same strand, a poly-lysine-conjugated DNA orRNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or thelike, that is not autonomously replicating. In many, but not all, commonembodiments, the vectors of the present invention are plasmids orbacmids.

In one embodiment, the genes encoding certain enzymes are cloned byexpression cloning (a.k.a. activity cloning). For example, the isolatedFusarium oxysporum strain MK7 is propagated, and cDNA libraries are madeusing polyadenine mRNA extracted from strain MK7. Expression vectors inthe cDNA libraries are then transformed into suitable host cells (e.g.,fungal cells, bacteria cells, yeast cells, plant cells, insect cells,and animal cells) and said host cells are screened for preferredenzymatic activity. In one embodiment, said enzymatic activity isselected from the group consisting of enzymatic activities of cellulase,xylanase, ligninase, glucuronidase, arabinofuranosidase,arabinogalactanase, ferulic acid esterase, lipase, pectinase,glucomannase, amylase, laminarinase, xyloglucanase, galactanase,glucoamylase, pectate lyase, chitinase, exo-β-D-glucosaminidase,cellobiose dehydrogenase, and acetylxylan esterase, xylosidase,α-L-arabinofuranosidase, feruloyl esterase, endoglucanase,β-glucosidase, Mn-peroxidase, and laccase.

In another embodiment, since the genome sequence of Fusarium oxysporumis available, a genomics/bioinformatics method can be taken to clonegenes encoding enzymes of interest. For example, data mining the genomesequence of Fusarium oxysporum for homologs of genes of interest canreveal candidate genes encoding protein with certain preferred enzymaticactivities. Then such genes can be cloned and tested for the activities.

Still in another embodiment, genes encoding protein with certainpreferred enzymatic activities can be isolated by traditional molecularcloning methods. For example, activities of certain proteins can beidentified from a culture bank, and said proteins can be purified. Thepurified enzymes are then subjected to analysis, and the peptidesequences can be determined. Degenerate primers are then designed, andused in a PCR or a RT-PCR to clone the genes of interest. To design thedegenerate primers, known homologous amino acid sequences can be alignedwith the protein of interest to identify conserved regions.

The isolated gene encoding an acid pH tolerant enzyme be cloned into anexpression vector, and transformed into a host cell selected from thegroup consisting of fungal cell (e.g., Fusarium species, Aspergillusspecies), bacteria cell (e.g., bacillus species), yeast cell, plantcell, insect cell, and animal cell. To select a suitable host cell,several factors need to be considered: the speed of cell growth, cost ofgrowth medium, expression levels, secretion capability, andpost-translational modifications (e.g., protein folding, N-linkedglycosylation, O-linked glycosylation, phosphorylation, acetylation andacylation).

In one embodiment, said gene encoding an acid pH tolerant enzyme istransformed into a fungal cell. Normally, a fungal strain which has (1)low secreted protease levels, (2) low total spectrum of secretedproteins, (3) capacity for high level heterologous expression, (4)favorable fermentation morphology, (5) “Generally Regarded As Safe”status, and (6) transformability, is preferred for expression. Methodsof transformation of filamentous fungi are well known in the art. In oneembodiment, the transformation is through bombardment (see, Aboul-Soudet al., Transformation of Fusarium oxysporum by particle bombardment andcharacterization of the resulting transformants expressing a GFPtransgene, January 2005, Mycopathologia, 158(4):475-482). In anotherembodiment, the transformation is Agrobacteria-mediated (see, Mullins etal., Agrobacterium-mediated transformation of Fusarium oxysporum: anefficient tool for insertional mutagenesis and gene transfer, 2001,Phytopathology, 91(2):173-180). In still another embodiment, saidtransformation is chemical-mediated (e.g., polyethylene glycol (PEG)mediated protoplast transformation, see, Powell et al., Journal ofBacteriology, June 1990, 172(6):3163-3171). For example, conidia(spores) of fungal host are germinated to make young germling myceliafirst. Subsequently, cell wall of mycelia is removed by lytic enzymemixture to form fungal protoplasts, which is osmotically fragile. Saidfungal protoplasts are mixed with DNA (encoding a selectable marker),CaCl₂ and PEG, and optionally, nuclease inhibitor (e.g.,aurintricarboxylic acid). Dilutions of the protoplasts were then spreadon osmotically-stabilized medium (e.g., minimal medium containingsucrose) to allow regeneration and selection of transformants A typicalexpression vector used in fungal host cell comprises a fungal promoter(which allows initiation of transcription in a fungal host), the gene ofinterest, a fungal terminator (which ensures termination oftranscription in a fungal host), and a selectable marker. Translationinitiation signals and DNA encoding signal peptide for secretion canalso be included. The selection marker can be selected from the groupconsisting of nutritional markers (e.g., argB (arginine prototrophy) andtrpC (trptophan prototrophy)), nutritional markers with forward andreverse selection (e.g., amdS (acetamidase), pyrG (uridine prototrophy),niaD (nitrate utilization), and sC (sulphate utilization)), and dominantselectable markers (e.g., benA (benomyl-resistant β-tubulin), oliC(oligomycin-resistant ATP synthase), hygB (hygromycin B resistance),G418 (geneticin resistance), phleomycin/bleomycin resistance, bar(confers resistance to BASTA (herbicide)). Non-limiting examples offungal expression vectors are pBARKS, pBARGEM, pBARMTE, pBARGP,pAn52-7Not uidA, pPFE2, p777, pAN, pTL, pUC19, and those described inCullen et al. (Molecular Cloning Vectors for Aspergillus and Neurospora,in A Survey of Molecular Cloning Vectors and Their Uses, ButterworthPublishers, Stoneham, M A 1986). Transformation of fungal hosts isintegrative, and is usually mitotically stable.

Fungal expression systems can be optimized. For example, major secretedhost proteins can be deleted so that the heterologous enzyme constitutesa high percentage of total protein. Genes encoding proteases andmucotoxins can also be deleted to stabilize expressed protein andunwanted side activity. In addition, constitutive/induced promoter,hotspots included in expression vectors, ectors, Kozak sequenceoptimization, manipulation of different binding sites in promoter canall be used.

The isolated fungal strain and/or its progeny of the present inventioncan be further mutagenized and screened for improved expression or forthe removal of one or more unwanted side-activities. Classicalmutagenesis methods can be applied, which include, but are not limitedto, chemical mutagenesis, insertional mutagenesis, and radiationmutagenesis (e.g., UV or gamma irradiation). In some cases, geneticcrosses can be done to combine improvements from different mutants.

Production of Biofuel and/or Precursors of Biofuel

Biofuels produced from sugar, starch, vegetable oil, or animal fatsusing conventional technology are called first-generation biofuels. Thebasic feedstocks for these biofuels are often seeds/grains containingstarch (e.g. wheat, corn) or vegetable oil (e.g. sunflower), which onlyrepresent small portions of whole plants. Due to the rising globalpopulation, feedstocks for producing biofuels have been criticized fordiverting food away from human and animal food chain, leading to foodshortages and price rises.

The second generation biofuel (e.g. cellulosic biofuels, biohydrogen,biomethanol, DMF, Bio-DME, Fischer-Tropsch diesel, biohydrogen diesel,mixed alcohols and wood diesel), which can be produced from non foodcrops, or non-edible parts of crops, including but not limited to, wastebiomass, stalks of wheat, corn, wood, and special biomass crops (e.g.Miscanthus), is more publicly and politically popular. Among these,cellulosic ethanol is a type of biofuel produced from lignocellulose, astructural material that comprises much of the biomass of plants. It iscomposed mainly of cellulose (31-49%), hemicellulose (16-26%) and lignin(19-26%). Lignin makes the plant rigid and resistant to compression andmust be removed before fermentation. Crop stover (e.g., leaves andstalks of corn, sorghum, soybean, et al.), Switchgrass, Miscanthus,woodchips and the byproducts of lawn and tree maintenance are some ofthe more popular cellulosic materials for ethanol production. Productionof cellulosic ethanol requires extra steps of processing raw materialsto release the sugar monomers for microorganisms fermentation fromlignin. Compared to first generation biofuel, lignocellulose-basedbiofuels have several advantages. Firstly, sources forlignocellulose-based biofuels are geographically more evenly distributedthan sugars/starch-based biofuels. Secondly, lignocellulosic rawmaterials minimize the potential conflict between land use for food (andfeed) production and energy feedstock production. The raw material isless expensive than conventional agricultural feedstock and can beproduced with lower input of fertilizers, pesticides, and energy.Thirdly, biofuels from lignocellulose generate low net greenhouse gasemissions, reducing environmental impacts, particularly climate change.

Generally, the feedstock will contain organic and inorganic nutrientsfor supporting the growth and metabolism of the isolated fungal strain.However, where necessary inorganic or organic nutrients are absent, orare present in insufficient amounts, the feedstock may be supplementedwith an aqueous phase containing said necessary inorganic or organicnutrients to support fungal growth and metabolism.

Bioalcohols

In one embodiment, the present invention provides methods of producingbioalcohols and/or precursors of bioalcohols from feedstocks using theisolated fungal strain of the present invention. Non-limiting exemplarybioalcohols produced are bioethanol, biomethanol, biobutanol,isobutanol, et al. In one embodiment, the bioalcohol is bioethanol.

In one embodiment, said bioalcohols are produced in fermentation underanaerobic conditions. In another embodiment, said bioalcohols areproduced in fermentation under substantially anaerobic conditions.

Non-limiting examples of feedstocks include, ligno-cellulosicfeedstocks, carbon containing waste products, carbohydrates, or acombination thereof. For example, ligno-cellulosic feedstocks areselected from the group consisting of agricultural crop residues (e.g.,wheat straw, barley straw, rice straw, small grain straw, corn stover,corn fibers (e.g., corn fiber gum (CFG), distillers dried grains (DDG),corn gluten meal (CGM)), switch grass, hay-alfalfa, sugarcane bagasse),non-agricultural biomass (e.g., algal mats, urban tree residue), forestproducts and industry residues (e.g., softwood first/secondary millresidue, hard softwood first/secondary mill residue, recycled paper pulpsludge), ligno-cellulosic containing waste (e.g., newsprint, wastepaper, brewing grains, used rubber tire (UTR), municipal organic waste,yard waste, clinical organic waste, waste generated during theproduction of biofuels (e.g. processed algal biomass, glycerol), and acombination thereof. The carbohydrates are selected from the groupconsisting of monosaccharides, disaccharides, oligosaccharides,polysaccharides, and mixture thereof. For example, the carbohydrates arefive and six carbon sugars.

Methods and strategies for the production of commodity biofuel and/orprecursors of biofuel and chemicals from feedstocks are known in theart. Major steps of producing cellulosic alcohols include pretreatment(making the lignocellulosic material such as wood or straw amenable tohydrolysis), cellulolysis (breaking down the molecules into sugars),separation (mainly isolating sugar solution from lignin), fermentationand distillation. Pretreatment techniques include acid hydrolysis, steamexplosion, ammonia fiber expansion, alkaline wet oxidation and ozonepretreatment (Klinke et al., 2004, Inhibition of ethanol-producing yeastand bacteria by degradation products produced during pre-treatment ofbiomass. Appl Microbiol Biotechnol 66:10-26.).

A fermenter (also called a bioreactor) is can be used as a container forthe production of biofuel. The first step of fermentation is tosterilize the fermenter. Sterilization can be done with steam,chemicals, washing, or combination of these. Numerous reactor designshave been reported, such as batch reactors, sequencing batch reactors,continuously stirred tank reactors, anaerobic contact reactors,anaerobic baffled reactors, fluidized-bed reactors, gas lift reactors,upflow anaerobic sludge blanket reactors and anaerobic hybrid reactors.Illustrative fermenters are those described in U.S. Pat. Nos. 3,615,253;5,205,936; 5,728,577; 4,530,762; 4,649,117; 7,446,156 and 5,228,995.According to different fermenters, there are three general ways in whichfermentations are done: batch fermentation, fed batch fermentation (orcontinuous fermentation) and cascade fermentation. For batchfermentation, the reactor is filled with sterile substrate andinoculated with fermentation organism. The culture is allowed to growuntil saturation. For fed batch fermentation (or continuousfermentation), substrate is fed continuously and the culture medium isremoved continuously. For cascade fermentation, the fermenting ‘liquor’is passed through a series of ferments to built up more and moreproducts (e.g. in brewing, the beer would be fermented in several stagesto increase the alcohol content.).

The fermentation is started with the inoculation of a small, activelygrowing sample of the microorganism to the fermenters (bioreactors)containing sterile substrate. Both prokaryotic microorganisms (includingbacteria, cyanobacteria) and eukaryotic microorganisms (including yeast,fungi and algae) have been used in industrial fermentation. In someother bioreactors, alternative organisms (including plant cells,mammalian cells) are used to produce products such as proteins,especially for pharmaceutical purposes. For instance, cell cultures ofseveral different plant species, including Arabidopsis thaliana, Taxuscuspidata, Catharathus roseus and important domestic crops such astobacco, alfalfa, rice, tomato and soybean, are used for recombinantprotein manufacture.

Microorganisms are used extensively to produce a great range of productsand service: such as traditional products (including but not limited to,bread, beer, wine, spirits, cheese, dairy products, fermented meats andvegetables, mushrooms, soy sauce and vinegar), agricultural products(including but not limited to, gibberellins, fungicides, insecticides,silage, amino acids such as L-Glutamine, L-Lysine, L-Tryptophan,L-Throenine, L-aspartic (+), L-arylglycines), enzymes (including but notlimited to, carbohydrates, celluloses, lipases, pectinases, proteases),fuels and chemical feedstocks (including but not limited to, acetone,butanol, butanediol, isopropanol, ethyl alcohol, glycerol, methane,glycerol, butyric acid, methane, citric acid, fumaric acid, lactic acid,propionic acid, succinic acid, itaconic acid, acetic acid,3-hydroxypropionic acid, glyconic acid, tartaric acid and L-glutaricacid or salts of any of these acids), nucleotides, organic acids,pharmaceuticals and related compounds (including but not limited to,alkaloids, antibiotics, hormones, immunosuppressant, interferon,steroids, vaccines, vitamins) and polymers (including but not limitedto, alginates, dextran, gellan, polyhydroxybutyrate, scleroglucan andxanthan).

Cellulolysis are currently performed by choosing from chemicalhydrolysis (e.g. U.S. Pat. Nos. 4,427,453; 4,556,430; 6,022,419),enzymatic hydrolysis (e.g. U.S. Pat. No. 5,637,502) and thermalhydrolysis (e.g. U.S. Pat. No. 6,692,578). Diluted acid is used underhigh heat and high pressure in the chemical hydrolysis (or concentratedacid at lower temperatures and pressure) to breakdown polysaccharidechains. Cellulase, xylanase and hemicellulase, which can be producedfrom fungi (Trichoderma reesei) at large amount (e.g. U.S. Pat. No.6,555,335), are used to convert biomass such as corn stover, distillergrains, wheat straw and sugar cane bagasse, or energy crops (e.g.Switchgrass) into fermentable sugars. In the fermentation step, baker'syeast (Saccharomyces cerevisiae) has been used in industrial productionof ethanol from hexoses (6-carbon sugar). Processes for the continuousfermentation of sugars to provide alcohol and ethanol fermentationprocesses featuring yeast recycle are known (viz., U.S. Pat. Nos.1,201,062; 2,054,736; 2,063,223; 2,122,939; 2,146,326; 2,169,244;2,230,318; 2,272,982; 2,285,130; 2,155,134; 2,371,208; 2,657,174;2,676,137; 2,967,107; 3,015,612; 3,078,166; 3,093,548; 3,177,005;3,201,328; 3,207,605; 3,207,606; 3,219,319; 3,234,026; 3,413,124;3,528,889; 3,575,813; 3,591,454; 3,658,647; 3,676,640; 3,705,841;3,737,323; 3,940,492; and, 3,984,286).

Ligno-cellosic materials comprise cellulose, hemicellulose and lignin.Cellulose is a polysaccharide comprising glucopyranose subunits joinedby β-1→4 glucosidic bonds. The monomer subunits are glucose.Hemicellulose are groups of polysaccharides including four basic types:D-xyloglucans, D-xylans, D-mannans, and D-galactans. In each type, twoto six monomers are linked by β-1→4 and β-1→3 bonds in main chained andα-1→2, 3, and 6 binds in branches. The monomer subunits can be D-xylose,L-arabinose, D-mannose, D-glucose, D-galactose, and D-glucouronic acid.Core lignins are highly condensed polymers formed by dehydrogenativepolymerization of the hydroxycinnamyl alcohols, p-coumaryl alcohols,coniferyl alcohols, and sinapyl alcohols. Non-core lignin includesesterified or etherified phenolic acids bound to core lignin ornoncellulosic polysaccharides. In preferred embodiments, the biomassmaterial comprising cellulose, hemicellulose and lignin (i.e.,lignocellulosic biomass) is treated to produce glucose, fructose,sucrose, mannose, maltose, sorbitol, galactose, xylose, and combinationsthereof.

In some embodiments, ligno-cellulosic biomass materials are hydrolyzedbefore fermentation. The present invention is not limited to the use ofany particular hydrolysis method. Indeed, the use of a variety ofhydrolysis methods are contemplated, including, but not limited to,enzymatic hydrolysis and chemical hydrolysis (such as dilute acidhydrolysis or concentrated acid hydrolysis) and combinations thereof.

Some examples of methods of pretreating feedstock are disclosed inpatent application publication nos. U.S. 2007/0161095, WO 05/053812, WO06/086757, U.S. 2006/0182857, U.S. 2006/177551, U.S. 2007/0110862, WO06/096834, WO 07/055735, U.S. 2007/0099278, WO 06/119318, U.S.2006/0172405, and U.S. 2005/0026262.

Examples of enzymes suitable for digestion of cellulose are disclosed inpatent or patent application publication nos. U.S. 2003/0096342, WO03/012109, U.S. Pat. No. 7,059,993, WO 03/012095, WO 03/012090, U.S.2003/0108988, U.S. 2004/0038334, U.S. 2003/0104522, EP 1 612 267, and WO06/003175.

The isolated fungal strain and/or its progeny of the present inventiondirectly can convert ligno-cellulosic feedstocks to energy-richmetabolites with few pretreatment steps and no enzyme additions.Consequently, use of this novel process is relatively simple andinexpensive in comparison to current methods. However, it is understoodthat the pretreatment steps and enzymes additions can still be employedas optional steps to increase hydrolysis rates and reduce productiontime of energy-rich metabolites.

Biodiesel and Biolubricant

Biodiesel refers to lipid-based diesel fuel consisting of long-chainalkyl (methyl, propyl or ethyl) esters, wherein the lipid comes fromliving or recently living organisms (e.g., vegetables oil, animal fat,microorganisms oil). Lipids may be employed as precursors in theproduction of biodiesels, for example, by transesterification. As usedherein, the term transesterification refers to the process of exchangingthe organic group R″ of an ester with the organic group R′ of analcohol:

These reactions are often catalyzed by the addition of an acid or base.Biodiesel is typically made by chemically reacting lipids (e.g.,vegetable oil, animal fat) with an alcohol. Non-limiting examples ofalcohols include, methanol, ethanol, butanol, isopropanol. Usingalcohols of higher molecular weights improves the cold flow propertiesof the resulting ester, at the cost of a less efficienttransesterification reaction. Any free fatty acids (FFAs) in the baseoil are either converted to soap and removed from the process, or theyare esterified (yielding more biodiesel) using an acidic catalyst. Aby-product of the transesterification process is the production ofglycerol.

Lipids as precursors of biodiesel can come from a lot of feedstocksources. Non-limiting examples of feedstocks include, rapeseed oil,soybean oil, field pennycress, jatropha, mustard, flax, sunflower, palmoil, coconut, hemp, castor oil, coconut oil, corn oil, cottonseed oil,peanut oil, radish oil, ramtil oil, rice bran oil, safflower oil,salicornia oil, sunflower oil, tung oil, algae oil, copaiba, honge oil,jatropha oil, jojoba oil, milk bush, petroleum nut oil, and animal fat.

Biodiesel is meant to be used in standard diesel engines and is thusdistinct from the vegetable and waste oils used to fuel converted dieselengines. Biodiesel can be used alone, or blended with petro-diesel.Blends of biodiesel and conventional hydrocarbon-based diesel areproducts most commonly distributed for use in the retail diesel fuelmarketplace. Blends of 20 percent biodiesel with 80 percent petroleumdiesel (B20) can generally be used in unmodified diesel engines.Biodiesel can also be used in its pure form (B100), but may requirecertain engine modifications to avoid maintenance and performanceproblems.

Biodiesel can also be used as a heating fuel in domestic and commercialboilers, a mix of heating oil and biofuel which is standardized andtaxed slightly differently than diesel fuel used for transportation. Itis sometimes known as “bioheat”. Heating biodiesel is available invarious blends; up to 20% biofuel is considered acceptable for use inexisting furnaces without modification.

Non-limiting examples of biodiesel production methods include, batchprocess, enzyme-free supercritical process, ultra- and high-shearin-line and batch reactor method, ultrasonic-reactor method andmicrowave method. More systems and apparatus for biodiesel productionare described in U.S. Pat. Nos. 7,452,515, 7,524,982, 7,420,072,6,824,682, 7,169,821, 6,979,426, 7,514,247, 7,449,313, 7,605,281, andU.S. Patent Application Nos. 20080282606, 20070260079, 20070010681,20090054701, 20030111410, 20080202021, 20050113467, 20050255013,20030175182, 20080299633, 20080102503, 20090178330, 20080313955,20080282687, 20070167642, and 20070122667, each of which is herebyincorporated by reference in its entirety.

Thus, the present invention provides methods of producing energy-richmetabolites using the isolated fungal strain of the present invention,where in the energy-rich metabolites are essentially lipids asprecursors of biodiesel. The lipids can be extracted from the isolatedfungal strains for production of biodiesel. The isolated fungal strainof the present invention has a more favorable lipid profile incomparison to algae and other lipid producing organisms. In oneembodiment, said lipids are essentially fatty acids. In one embodiment,said fatty acids are essentially unsaturated fatty acids and/orsaturated fatty acids. In one embodiment, said unsaturated fatty acidsare selected from the group consisting of oleic acid (18:1), α-linolenicacid (18:3), eicosenoic acid (20:1), and a combination thereof. In oneembodiment, said saturated fatty acids are selected from the groupconsisting of palitic acids (16:0), stearic acids (18:0), arachidic acid(20:0), behenic acid (22:0), and a combination thereof. Other types oflipids that may be produced include, but are not limited to, saturatedfats (e.g., butyric acid, hexanoic acid, octanoic acid, decanoic acid,dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoicacid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid,nonadecanoic acid, eicosanoic acid, docosanoic acid, tetracosanoicacid), Monounsaturated fats (e.g., tetradecenoic acid, pentadecenoicacid, hexadecenoic acid, heptadecenoic acid, octadecenoic acid,eicosenoic acid, docosenoic acid, cis-tetracosenoic acid), andPolyunsaturated fats (e.g., hexadecadienoic acid, linoleic acid,linolenic acid, acid, gamma-linolenic acid, parinaric acid,eicosadienoic acid, arachidonic acid, timnodonic acid, brassic acid,clupanodonic acid and docosahexaenoic acid), omega-7 vaccenic acid(Methyl 11-octadecenoate), omega-7 palmitoleic acid (methylhexadec-9-enoate; trade name Provinal™) and tetracosanoic acid, methylester, and other fatty acids listed in Table 1.

In one embodiment, said lipids as precursors of biodiesel are producedin fermentation under aerobic conditions. In another embodiment, saidbioalcohols are produced in fermentation under substantially aerobicconditions.

In one embodiment, the isolated fungal strain and/or its progeny iscapable of efficient production of the lipids. In some embodiments ofthe present invention, the amount of lipids produced is at least about 1g/L/day, 5 g/L/day, at least about 10 g/L/day, at least about 20g/L/day, at least about 30 g/L/day, at least about 40 g/L/day, at leastabout 50 g/L/day, at least about 60 g/L/day, at least about 70 g/L/day,or more. For example, the amount of biological oil produced is fromabout 1 g/L/day to about 5 g/L/day, from about 5 g/L/day to about 70g/L/day, from about 10 g/L/day to about 70 g/L/day, from about 20g/L/day to about 70 g/L/day, or from about 30 g/L/day to about 70g/L/day.

Lipids in a sample can be extracted using different procedures.Non-limiting examples of lipids extraction are described in King et al.(Supercritical Fluid Extraction: Present Status and Prospects, 2002,Grasa Asceites, 53, 8-21), Folch et al. (A simple method for theisolation and purification of total lipids from animal tissues, 1957, J.Biol. Chem., 226, 497-509), Bligh and Dyer (A rapid method of totallipid extraction and purification. 1959, Can. J. Biochem. Physiol., 37,911-917), Cabrini et al. (Extraction of lipids and lipophilicantioxidants from fish tissues—a comparison among different methods.1992, Comp. Biochem. Physiol., 101B, 383-386), Hara et al. (Lipidextraction of tissues with a low toxicity solvent. 1978, Anal. Biochem.,90, 420-426), Lin et al. (Ethyl acetate/ethyl alcohol mixtures as analternative to Folch reagent for extracting animal lipids. 2004, J.Agric. Food Chem., 52, 4984-4986), Whiteley et al. (Lipid peroxidationin liver tissue specimens stored at subzero temperatures. 1992,Cryo-Letters, 13, 83-86), Kramer et al. (A comparison of procedures todetermine free fatty acids in rat heart. 1978, J. Lipid Res., 19,103-106) and Somashekar et al. (Efficacy of extraction methods for lipidand fatty acid composition from fungal cultures, 2001, World Journal ofMicrobiology and Biotechnology, 17(3): 317-320). In another example,lipid can be extracted by methods similar to the FRIOLEX® (WestfaliaSeparator Industry GmbH, Germany) process is used to extract thebiological oils produced by the microorganisms. FRIOLEX® is awater-based physical oil extraction process, whereby raw materialcontaining oil can be used directly for extracting oil without using anyconventional solvent extraction methods. In this process, awater-soluble organic solvent can be used as a process aid and the oilis separated from the raw material broth by density separation usinggravity or centrifugal forces.

After the lipids have been extracted, the lipids can be recovered orseparated from non-lipid components by any suitable means known in theart. For example, low-cost physical and/or mechanical techniques areused to separate the lipid-containing compositions from non-lipidcompositions. If multiple phases or fractions are created by theextraction method used to extract the lipids, where one or more phasesor fractions contain lipids, a method for recovering thelipid-containing phases or fractions can involve physically removing thelipid-containing phases or fractions from the non-lipid phases orfractions, or vice versa. In some embodiments of the present invention,a FRIOLEX® type method is used to extract the lipids produced by themicroorganisms and the lipid-rich light phase is then physicallyseparated from the protein-rich heavy phase (such as by skimming off thelipid-rich phase that is on top of the protein-rich heavy phase afterdensity separation).

In some embodiments of the present invention, the ligno-cellulosicfeedstock that is used to grow the isolated fungal strain comprisescellulose in an amount of from about 5% to about 100%, from about 10% toabout 95%, from about 20% to about 90%, from about 30% to about 85%,from about 40% to about 80%, from about 50% to about 75%, or from about60% to about 70% by dry weight of the carbon feedstock. In someembodiments of the present invention, the cellulosic feedstock comprisescellulose in an amount of at least about 5%, at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, or at least about 70% of the dry weight of thecarbon feedstock. In some embodiments of the present invention, thecellulosic feedstock used to grow the isolated fungal strain comprisesfrom about 1% to about 50%, from about 5% to about 40%, or from about10% to about 30% by weight of a component selected from lignin,hemicellulose, or a combination thereof. In some embodiments of thepresent invention, the cellulosic feedstock used to grow a microorganismcomprises at least about 1%, at least about 5%, at least about 10%, atleast about 20%, or at least about 30% by weight of a component selectedfrom lignin, hemicellulose, or a combination thereof.

There are at least two stages in the production of lipids using theisolated fungal strain: biomass accumulation stage and lipid productionstage. In some embodiments of the present invention, the biomassaccumulation stage produces biomass of the fungal strain such that about10% to about 95%, about 20% to about 95%, about 30% to about 95%, about40% to about 95%, or about 50% to about 95% of the total biomassproduction of the fungal strain is achieved during the biomassaccumulation stage. In further embodiments of the present invention,about 60% to about 95%, about 70% to about 95%, or about 80% to about95% of the total biomass production of the microorganism is achievedduring the biomass accumulation stage. In some embodiments of thepresent invention, the biomass accumulation stage produces biomass ofthe microorganism such that at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, or atleast about 95% of the total biomass production of the microorganism isachieved during the biomass accumulation stage. For example, about 50%to about 95% of the total biomass production of the microorganism isachieved during the biomass accumulation stage. In some embodiments ofthe present invention, the lipid accumulation stage produces lipids suchthat about 10% to about 95%, about 20% to about 95%, about 30% to about95%, about 40% to about 95%, or about-50% to about 95% of the totallipid production of the microorganism is achieved during the lipidaccumulation stage. In further embodiments of the present invention,about 60% to about 95%, about 70% to about 95%, or about 80% to about95% of the total lipid production of the microorganism is achievedduring the lipid accumulation stage. In some embodiments of the presentinvention, the lipid accumulation stage produces lipids such that atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, or at least about 95% of the totallipid production of the microorganism is achieved during the lipidaccumulation stage. Preferably, about 50% to about 95% of the totallipid production of the microorganism is achieved during the lipidaccumulation stage.

Once the lipids are produced in accordance with the present invention,various methods known in the art can be used to transform the biologicaloils into esters of fatty acids for use as biodiesel, jet biofuel, or asingredients for food or pharmaceutical products. In some embodiments ofthe present invention, the production of esters of fatty acids comprisestransesterifying the biological oils produced by the microorganism. Insome embodiments of the present invention, the extraction of the lipidsfrom the microorganisms and the transesterification of the lipids can beperformed simultaneously, in a one step method. For example, the culturecontaining the isolated fungal strain can be exposed to conditions ortreatments (or a combination of conditions or treatments) that promoteboth extraction of the lipids and the transesterification of the lipids.Such conditions or treatments could include, but are not limited to, pH,temperature, pressure, the presence of solvents, the presence of water,the presence of catalysts or enzymes, the presence of detergents, andphysical/mechanical forces. Two sets of conditions or treatments couldbe combined to produce a one step method of extracting andtransesterifying the lipids, where one set of conditions or treatmentsfavorably promotes extraction of the lipids and the other set ofconditions or treatments favorably promotes transesterification of thelipids, so long as the two sets of conditions or treatments can becombined without causing significant reduction in the efficiency ofeither the extraction or the transesterification of the lipids. In someembodiments of the present invention, hydrolysis and transesterificationcan be performed directly of whole-cell biomass. In other embodiments ofthe present invention, the extraction of the lipids is performed as astep that is separate from the step of transesterification of thelipids. In one embodiment, such transesterification reactions areperformed using acid or base catalysts. In some embodiments of thepresent invention, methods for transesterifying the biological lipidsinto esters of fatty acids for use as biodiesel or as ingredients forfood or pharmaceutical products involve reacting the biological oilscontaining triglycerides in the presence of an alcohol and a base toproduce esters of the fatty acid residues from the triglycerides.

Alcohols suitable for use in transesterification include any lower alkylalcohol containing from 1 to 6 carbon atoms (i.e., a C 1-6 alkylalcohol, such as methyl, ethyl, isopropyl, butyl, pentyl, hexyl alcoholsand isomers thereof). Without being bound by theory, it is believed thatin some embodiments of the present invention, the use of lower alkylalcohols in the methods of the present invention produces lower alkylesters of the fatty acid residues. For example, the use of ethanolproduces ethyl esters. In certain embodiments, the alcohol is methanolor ethanol. In these embodiments, the fatty acid esters produced are amethyl ester and an ethyl ester of the fatty acid residue, respectively.In processes of the present invention, the alcohol typically comprisesfrom about 5 wt. % to about 70 wt. %, from about 5 wt. % to about 60 wt.%, from about 5% to about 50 wt. %, from about 7 wt. % to about 40 wt.%, from about 9 wt. % to about 30 wt. %, or from about 10 wt. % to about25 wt. % of the mixture of the lipids composition, the alcohol and thebase. In certain embodiments, the composition and the base can be addedto either pure ethanol or pure methanol. In general, the amount ofalcohol used may vary with the solubility of the lipids or compositioncontaining triglycerides in the alcohol.

The composition comprising triglycerides, the alcohol and the base arereacted together at a temperature and for an amount of time that allowsthe production of an ester from the fatty acid residues and the alcohol.Suitable reaction times and temperatures may be determined by one ofskill in the art to produce an ester. Without intending to be bound bytheory, the fatty acid residues are believed to be cleaved from theglycerol backbone of the triglyceride and esters of each fatty acidresidue are formed during the step of reacting. In certain embodiments,the step of reacting the composition in the presence of an alcohol and abase is performed at a temperature from about 20° C. to about 140° C.,from about 20° C. to about 120° C., from about 20° C. to about 110° C.,from about 20° C. to about 100° C., or from about 20° C. to about 90° C.In further embodiments, the step of reacting the composition in thepresence of an alcohol and a base is performed at a temperature of atleast about 20° C., 75° C., 80° C., 85° C., 90° C. 95° C., 105° C., or120° C. In some embodiments of the present invention, the step ofreacting the composition in the presence of an alcohol and a base isperformed at a temperature of about 20° C., 75° C., 80° C., 85° C., 90°C., 95° C., 105° C., or 120° C. In some embodiments, the step ofreacting the composition in the presence of an alcohol and a base isperformed for a time from about 2 hours to about 36 hours, from about 3hours to about 36 hours, from about 4 hours to about 36 hours, fromabout 5 hours to about 36 hours, or from about 6 hours to about 36hours. In certain embodiments, the step of reacting the composition inthe presence of an alcohol and a base is performed for about 0.25, 0.5,1.0, 2.0, 4.0, 5.0, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 10, 12, 16, 20, 24, 28,32, or 36 hours.

In one embodiment, the step of reacting the lipids composition, alcoholand base may be conducted by refluxing the components to produce thefatty acid esters, such as PUFA esters. In additional embodiments, thestep of reacting the lipids composition may be carried out at atemperature that does not result in the refluxing of the reactioncomponents. For example, carrying out the step of reacting the lipidscomposition under pressures greater than atmospheric pressure canincrease the boiling point of the solvents present in the reactionmixture. Under such conditions, the reaction can occur at a temperatureat which the solvents would boil at atmospheric pressure, but would notresult in the refluxing of the reaction components. In some embodiments,the reaction is conducted at a pressure from about 5 to about 20 poundsper square inch (psi); from about 7 to about 15 psi; or from about 9 toabout 12 psi. In certain embodiments, the reaction is conducted at apressure of 7, 8, 9, 10, 11, or 12 psi. Reactions conducted underpressure may be carried out at the reaction temperatures listed above.In some embodiments, reactions conducted under pressure may be carriedout at a temperature of at least about 70° C., 75° C., 80° C., 85° C.,or 90° C. In some embodiments, reactions conducted under pressure may becarried out at 70° C., 75° C., 80° C., 85° C., or 90° C.

In one embodiment of the present invention, fatty acid esters areseparated from the reaction mixture by distilling the composition torecover a fraction comprising the ester of the fatty acid. A targetedfraction of the reaction mixture including the fatty acid esters ofinterest can be separated from the reaction mixture and recovered. Incertain embodiments, the distillation is performed under vacuum. Withoutbeing bound by theory, distillation under vacuum allows the distillationto be accomplished at a lower temperature than in the absence of avacuum and thus may prevent the degradation of the esters. Typicaldistillation temperatures range from about 120° C. to about 170° C. Insome embodiments, the step of distilling is performed at a temperatureof less than about 180° C., less than about 175° C., less than about170° C., less than about 165° C., less than about 160° C., less thanabout 155° C., less than about 150° C., less than about 145° C., lessthan about 140° C., less than about 135° C., or less than about 130° C.Typical pressures for vacuum distillation range from about 0.1 mm Hg toabout 10 mm Hg. In some embodiments, the pressure for vacuumdistillation is at least about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 mmHg. In some embodiments of the present invention, the pressure forvacuum distillation is about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4mmHg.

Renewable oils, waxes and fatty acids have significant demand in a widerange of industries (e.g. neutraceuticals, biolubricants, cosmetics,candles and soaps) and command a premium price compared to petroleumbased products. High profit margins are usually expected for the lipidproducts, which can sell for greater than $2.00/pound, equating to $600revenue per ton of feedstock (wheat straw costs $60/ton). Consumers willclearly benefit from this technology through the reduction of theirdependence on petroleum-based products while generating a market foragricultural wastes and by-products. Since few companies and researchgroups are investigating fungal lipid production directly fromlignocellulosic biomass for higher value products, both industry and thescientific community will benefit from the knowledge gained by thisinvention.

The renewable lipids are ideal for the increasing number of consumersdemanding carbon-neutral, natural biobased products, which are currentlyproduced primarily from limited plant and animal resources. The processof the invention offers an alternative to these renewable lipid sourcesand creates a market for waste agricultural feedstocks. The process ofthe invention is further developed for commercial production of lipidproducts from waste organic feedstocks. The process of the invention isoptimized with a bench pilot-scale system, performing process flowdesign and techno-economic analysis of commercial production systems,analyzing lipid products in detail, and targeting appropriatemarkets/customers for the products. A pilot level demonstration systemis designed to produce economically viable yields of high-value lipidsfrom a variety of substrates for defined target markets and customers.

In another embodiment, the lipids extracted from the isolated fungalstrain and/or its progeny of the present invention are used to producebiolubricants. As used herein, the term “biolubricants” refers tolubricants produced by using material originated from living or recentlyliving organisms. As used herein, the term “lubricants” refers tosubstances (usually a fluid under operating conditions) introducedbetween two moving surfaces so to reduce the friction and wear betweenthem. Base oils used as motor oils are generally classified by theAmerican Petroleum Institute as being mineral oils (Group I, II, andIII) or synthetic oils (Group IV and V). See American PetroleumInstitute (API) Publication Number 1509. One of the single largestapplications for lubricants, in the form of motor oil, is to protect theinternal combustion engines in motor vehicles and powered equipment.Typically lubricants contain 90% base oil (most often petroleumfractions, called mineral oils) and less than 10% additives. Vegetableoils or synthetic liquids such as hydrogenated polyolefins, esters,silicones, fluorocarbons and many others are sometimes used as baseoils. These are primarily triglyceride esters derived from plants andanimals. For lubricant base oil use the vegetable derived materials arepreferred. Common ones include high oleic canola oil, castor oil, palmoil, sunflower seed oil and rapeseed oil from vegetable, and Tall oilfrom animal sources. Many vegetable oils are often hydrolyzed to yieldthe acids which are subsequently combined selectively to form specialistsynthetic esters.

Additives deliver reduced friction and wear, increased viscosity,improved viscosity index, resistance to corrosion and oxidation, agingor contamination, etc. Lubricants such as 2-cycle oil are also added tosome fuels. Sulfur impurities in fuels also provide some lubricationproperties, which have to be taken in account when switching to alow-sulfur diesel; biodiesel is a popular diesel fuel additive providingadditional lubricity. Non-liquid lubricants include grease, powders (drygraphite, PTFE, Molybdenum disulfide, tungsten disulfide, etc.), teflontape used in plumbing, air cushion and others. Dry lubricants such asgraphite, molybdenum disulfide and tungsten disulfide also offerlubrication at temperatures (up to 350° C.) higher than liquid andoil-based lubricants are able to operate. Limited interest has beenshown in low friction properties of compacted oxide glaze layers formedat several hundred degrees Celsius in metallic sliding systems, however,practical use is still many years away due to their physically unstablenature. Another approach to reducing friction and wear is to usebearings such as ball bearings, roller bearings or air bearings, whichin turn require internal lubrication themselves, or to use sound, in thecase of acoustic lubrication. In addition to industrial applications,lubricants are used for many other purposes. Other uses includebio-medical applications (e.g. lubricants for artificial joints) and theuse of personal lubricant for sexual purposes.

Thus, the lipids extracted from the culture of the isolated fungalstrain and/or its progeny of the present invention can be used toproduce ester-based biolubricant compositions, by adding suitableadditives. Methods of making ester-based lubricant compositions areknown to one skilled in the art. For a non-limiting example, a quantityof biologically-derived oil comprising triglycerides is provided andprocessed so as to hydrolyze at least some of the triglycerides and formfree fatty acids, wherein the fatty acids are of a type selected fromthe group consisting of saturated fatty acids, monounsaturated fattyacids, and polyunsaturated fatty acids, and combinations thereof. Thefatty acids are separated by type, such that at least themonounsaturated fatty acids are substantially isolated from thesaturated fatty acids and the polyunsaturated fatty acids. Next, atleast some of the monounsaturated fatty acids are modified to form anester product (e.g., comprising triesters), and at least some of thesaturated fatty acids and/or polyunsaturated fatty acids arehydrotreated to yield alkanes (paraffins). Note also that in someembodiments, such ester products can include one or more of thefollowing: mono-, di-, and triester species, and hydroxylated analoguesthereof.

Biohydrogen

The term biohydrogen here refers to hydrogen produced via biologicalprocesses, for example, via fermentation using the isolated fungalstrain and/or its progeny of the present invention. The presentinvention provides methods of producing energy-rich metabolites usingthe isolated fungal strain and/or its progeny of the present invention,wherein the energy-rich metabolites are biogas, for example, hydrogen ormethane. In one embodiment, hydrogen is produced under anaerobicconditions. In another embodiment, hydrogen is produced undersubstantially anaerobic conditions.

In one embodiment, feedstocks are mixed with the isolate fungal strainand/or its progeny of the present invention in a bioreactor to producehydrogen. To accelerate degradation of feedstocks, vacuum can be used,for example, at about 0.1 atm, about 0.2 atm, about 0.3 atm, about 0.4atm, about 0.5 atm, to remove gases including hydrogen out of thebioreactor. In one embodiment, production of other biogas, such asmethane is inhibited to increase the production of hydrogen.Non-limiting examples systems and bioreactors of producing hydrogen aredescribed in U.S. Pat. Nos. 7,473,552 and 7,083,956, which are herebyincorporated by reference in their entireties.

Biofuels from Sludge Materials

The isolated strain and/or its progeny of the present invention istolerant to high concentration of metals and extreme pH conditions.Thus, the present invention provides methods for producing biofueland/or precursors of biofuels using starting raw material, municipal,industrial, and/or farm sewage sludge or waste as carbon-containingfeedstocks. The sludge may be treated municipal or industrial raw sludgeor primary solids, or treated farm sludge. That is, the sludge in someembodiments has undergone one or more treatment processes such asanaerobic and/or aerobic digestion, composting, and/or at least onechemical or physical processing such as drying, dewatering, thickening,pressing, filtering, centrifugation, ultraviolet or chemicaldisinfection (e.g., chlorine disinfection), lime stabilization, and/orthermal processing.

In some embodiments, the sludge is a recalcitrant sludge having a highcontent of heavy metals such as one or more of Mn, Zn, Pb, Cu, Cr, Ni,Cd, and Hg. At least one, two, or three such heavy metal(s) (or heavymetals taken collectively) may be present at more than 10 ppm, or 50ppm, or 100 ppm, or 200 ppm, or 400 ppm, or 500 ppm in the recalcitrantsludge, by weight. At least one such heavy metal (or heavy metals takencollectively) may be present at from about 400 to about 1000 ppm. Forexample, the recalcitrant sludge may be contaminated at such levels withlead (Pb) and/or cadmium (Cd). In one embodiment, said sludge or wasteis acidic. In one embodiment, the pH of the sludge or waste ranges fromabout 0 to about 8, for example, from about 0.7 to about 7.5.

In another embodiment, said heavy metals in the sludge or waste arerecovered through biosorption by the isolated fungal strain of thepresent invention, and said acidic sludge or waste is neutralized.

The isolated fungal strain and/or its progeny are also resistant tocontaminations due to antibiotics produced inside (e.g., naphthazarinpigments). Thus, in these or other embodiments, the sludge has a highcontent of at least one bacterial, viral, and/or parasitic pathogen,including a variety of enteric pathogens, for example, enteropathogenicE. coli, Salmonella, Shigella, Yersinia, Vibrio Cholerae,Cryptosporidium, Giardia, Entamoeba, Norovirus, and Rotavirus, amongothers.

In certain embodiments, the sludge is generated by industrialactivities. That is, the sludge is an end product of a waste watertreatment plant of an industrial facility, such as a chemical,pharmaceutical, or paper production facility, or food processingfacility.

The sludge may originate from a pharmaceutical production facility. Inaccordance with the invention, so long as the compounds produced by thepharmaceutical production facility are suitable feedstock for theisolated fungi strain, they can may be degraded by microbial metabolismand converted to biofuels.

The sludge may originate from a paper production facility. The pulp andpaper industry is responsible for large discharges of highly pollutedeffluents. These pollutants, whose main characteristics are their hightoxicity and low biodegradability, include a variety of tannins,lignins, resins, terpenes, and chlorophenolic compounds. The compositionof these effluents, which has a great influence on its treatability, mayvary considerably, depending on the raw material and manufacturingprocess. The present invention, however, provides methods and systemsthat are versatile with regard to synthesizing biofuel components fromthese toxic effluents.

In some embodiments, the sludge is generated by a food processingfacility.

In some embodiments, the sludge is farm sludge comprising animal manure.Most animal manure is feces. Common forms of animal manure include FYM(farmyard manure) or farm slurry (liquid manure). Farmyard manure mayalso comprise plant material (often straw) which has been used asbedding for animals and has absorbed the feces and urine. Agriculturalmanure in liquid form is known as slurry, and is produced by moreintensive livestock rearing systems where concrete or slats are used,instead of straw bedding.

Thus, the present invention provides methods of producing one or moreenergy-rich metabolites as biofuel or precursors of biofuel using one ormore said isolated fungus stains as described above, comprising:

-   -   a) making a mixture of one or more said isolated fungal strains        and/or its progeny with a feedstock material selected from the        group consisting of ligno-cellulosic feedstocks, carbon        containing waste products, carbohydrates, and a combination        thereof in a container, wherein the material can support the        growth of said isolated fungal strain and/or its progeny;    -   b) growing said isolated fungal strain in said mixture to        produce one or more biofuels and/or precursors of biofuel; and    -   c) optionally, isolating said biofuels and/or precursors of        biofuel from the mixture.

In one embodiment, additional compounds necessary for the growth of theisolated fungal strain and/or its progeny of the present invention areadded into the mixture wherein the feedstocks do not contain, or containinsufficient mounts of said compounds necessary for the growth of thefungal strain. The isolated fungal strain and/or its progeny of thepresent invention can be used for biofuel and/or precursors of biofuelproduction from varies sources, such as ligno-cellulosic feedstocks,carbon containing waste products, carbohydrates, sugar monomers, or acombination thereof. In one embodiment, said biofuel is bioalcohol,biohydrogen, and/or biodiesel. In one embodiment, said precursors ofbiofuel are lipids or sugars. In one embodiment, said feedstocks areselected from the group consisting of ligno-cellulosic (e.g., algalmats, wheat straw, barley straw, corn stover, switch grass, spentbrewing grains, forest products, energy crop material), carbohydrates(e.g., monosaccharides, disaccharides, oligosaccharides,polysaccharides), waste (e.g., municipal, industrial, and farm sewagesludge), and combination thereof.

The ligno-cellulosic feedstocks can be selected from the groupconsisting of agricultural crop residues (e.g., wheat straw, barleystraw, rice straw, small grain straw, corn stover, corn fibers (e.g.,corn fiber gum (CFG), distillers dried grains (DDG), corn gluten meal(CGM)), switch grass, hay-alfalfa, sugarcane bagasse), non-agriculturalbiomass (e.g., algal mats, urban tree residue), forest products andindustry residues (e.g., softwood first/secondary mill residue, hardsoftwood first/secondary mill residue, recycled paper pulp sludge),ligno-cellulosic containing waste (e.g., newsprint, waste paper, brewinggrains, used rubber tire (UTR), municipal organic waste, yard waste,clinical organic waste, waste generated during the production ofbiofuels (e.g. processed algal biomass, glycerol), and a combinationthereof.

The carbon containing waste products can be selected from the groupconsisting of municipal organic waste, yard waste, industrial facilitywaste, such as waste from a chemical, pharmaceutical, or paperproduction facility, or food processing facility, and a combinationthereof.

Carbohydrates feedstocks can be selected from the group consisting ofmonosaccharides, disaccharides, oligosaccharides, polysaccharides, and acombination thereof. In one embodiment, said sugar monomers are selectedfrom the group consisting of trioses, tetroses, pentoses, hexoses,heptoses, et al., and a combination thereof. In one embodiment, saidpentoses are selected from the group consisting of ribulose, xylulose,ribose, arabinose, xylose, lyxose, deoxyribose, and a combinationthereof. In one embodiment, said hexoses are selected from the groupconsisting of allose, altrose, glucose, mannose, glucose, idose,galactose, talose, psicose, fructose, sorbose, tagatose, and acombination thereof.

In one embodiment, said mixture further comprises at least one componentselected from the group consisting of acidification materials, manganesedonors, and nutrient additions, pH buffering materials.

In one embodiment, said mixture has an initial pH 0.5 to 7.5 and willincrease during fermentation unless in a buffered media or culturevessel with pH control from about 0 to about 8.0, for example, fromabout 0.5 to about 7.5, from about 0.5 to about 3.0 or from about 3.0 toabout 7.0.

The methods of producing energy-rich metabolites using the isolatedfungal strain and/or its progeny of the present invention have otheradvantages. For example, the present invention provides novel methods ofproducing energy-rich metabolites as biofuel or precursors of biofuelfrom feedstocks such as ligno-cellulosic materials (e.g., wood orstraw). Currently existing methods of biofuel production fromligno-cellulosic materials require various expensive pretreatments andenzyme additions. One advantage of the present invention is that it doesnot require pretreatment steps of feedstocks, such as making thelignocellulosic material (e.g., wood or straw) amenable to hydrolysis,or cellulolysis, and enzymes additions, because the isolated fungalstrain of the present invention itself can perform these steps. Thus,the process described here directly converts ligno-cellulosic feedstocksto energy-rich metabolites with few pretreatment steps and no enzymeadditions. Consequently, use of this novel process is relatively simpleand inexpensive in comparison to current methods. For another example,the isolated fungus of the present invention can be used to produceenergy-rich metabolites as biofuel or precursors of biofuel over a widepH range, thus, negating the need for costly pH neutralization steps.For another example, in the production of lipids as precursors ofbiodiesel, the isolated fungal strain of the present invention has amore favorable lipid profile in comparison to algae and other lipidproducing organisms. For still another example, the isolated fungalstrain and/or its progeny of the present invention is highly resistantto contamination by other organisms, since members of the Fusarium genusare known to generate naphthazarinoid pigments, which have potentantibiotic, insecticidal and phytotoxic properties (Brimble et al.,1999). Thus, the methods of producing energy-rich metabolites of thepresent invention do not require the use of costly and time consumingmethods to prevent contamination as other currently microbial basedproduction of biofuels. For still another example, the isolated fungalstrain and/or its progeny of the present invention is tolerant to highconcentration of metals, such as Mn, which can be used to increaselignin degradation rates.

Cultures and Compositions Comprising the Isolated Fungal Strain

Fusarium oxysporum strain MK7 is a new strain of acidophilic fungus,which can directly convert ligno-cellulosic feedstocks, carbohydrates(e.g., 5 and 6 carbon sugars), and biomass (e.g., algal biomass) toenergy-rich substrates including lipids, ethanol and hydrogen.

The present invention provides a biologically pure culture having one,two, more or all of the identifying characteristics of the isolatedfungal strain of Fusarium oxysporum and/or its progeny as describedherein. In one embodiment, the biologically pure culture comprises theisolated fungal strain of Fusarium oxysporum, designated as MK7 whichhas been deposited as ATCC Accession Deposit No. PTA-10698, or activemutants thereof. In one embodiment, said isolated fungal strain and/orits progeny are in the form of conidia, pycnidia, chlamydospores,fragments of hyphae, or a combination thereof.

Applicant has made a deposit on behalf of National Park Service,Yellowstone National Park, USA on Mar. 2, 2010, of 25 vials of Fusariumoxysporum strain MK7 (as described herein) under the Budapest Treatywith the American Type Culture Collection (ATCC), P.O. Box 1549,Manassas, Va. 20108 USA, ATCC Accession No. PTA-10698. The strain wasdeposited prior to the filing date of this application. To satisfy theenablement requirements of 35 U.S.C. 112, and to certify that thedeposit of the isolated strain of the present invention meets thecriteria set forth in 37 CFR 1.801-1.809, Applicants hereby make thefollowing statements regarding the deposited Fusarium oxysporum strainMK7 (deposited as ATCC Accession Deposit No. PTA-10698):

1. During the pendency of this application, access to the invention willbe afforded to the Commissioner upon request;

2. Upon granting of the patent the strain will be available to thepublic under conditions specified in 37 CFR 1.808; 3. The deposit willbe maintained in a public repository for a period of 30 years or 5 yearsafter the last request or for the enforceable life of the patent,whichever is longer;

4. The viability of the biological material at the time of deposit willbe tested; and,

5. The deposit will be replaced if it should ever become unavailable.

The present invention further provides a composition comprising anisolated fungal strain of Fusarium oxysporum having at least one, two,more or all of the identifying characteristics as described herein. Inone embodiment, said isolated fungal strain and/or its progeny are inthe form of conidia, pycnidia, chlamydospores, fragments of hyphae, or acombination thereof. In some embodiments, the composition furthercomprises one or more components selected from the group consisting of amedium that supports growth of the fungal strain, an acidificationmaterial, a manganese donor, a nutrient addition, and a mixture thereof.In one embodiment, the medium is a solid. In another embodiment, themedium is a liquid.

In one embodiment, the composition comprises a medium supporting thegrowth of the isolated fungal strain and/or its progeny of the presentinvention. Suitable medium for isolating, growing, and maintainingFusarium oxysporum are well known by those skilled in the art.Non-limiting examples of such media for Fusarium oxysporum are describedin Gunner et al (1964, Anaerobic Growth of Fusarium oxysporum, J.Bacteriol. 87:1309-1316), Joshi et al (1987, The influence of variouscarbon and nitrogen sources on oil production by Fusarium oxysporum,32(2):124-129), De La Broise et al (1989, Osmotic, biomass, and oxygeneffects on the growth rate of Fusarium oxysporum using adissolved-oxygen-controlled turbidostat, Biotechol. Bioeng.33(6):699-705), and Norio et al (2007, Selected media for Fusariumoxysporum, Journal of General Plant Pathology, 73(5):342-348; 2002,Selective medium for Fusarium oxysporum and nitrate metabolic capacitydefective strain, Kyushu Okinawa Nogyo Kenkyu Seika Joho, 17:491-492),each of which is hereby incorporated by reference in its entirety.

The nutrition additions can be selected from the group consisting ofcarbohydrates (e.g., monosaccharides, polysaccharides), amino acidsdonor (e.g., amino acid, polypeptides), micronutrients (e.g., calcium,ammonium, copper, potassium, sodium, borax, ferrous, zinc, et al.), andcombination thereof.

Acid pH Tolerant Enzymes from F. oxysporum Strain MK7

The current technology for ethanol production from ligno-cellulosicmaterials requires several steps prior to fermentation by yeast. Thesesteps may include thermal degradation, dilute or concentrated acidhydrolysis, alkaline hydrolysis, or oxidation by chemicals such ashydrogen peroxide (Hendricks and Zeeman, 2009, Pretreatments to enhancethe digestibility of lignocellulosic biomass. Bioresource Technology,100:10-18.). The goal of pretreatment is to solubilize hemicellulose andlignin and decrystallize cellulose. However, before fermentation byyeast, the mixture must be pH neutralized and treated with enzymes todepolymerize cellulose into glucose.

The genome of Fusarium oxysporum f sp. lycopersici strain 4287 hasrecently been sequenced and has been shown to carry a variety of genesinvolved in the degradation of lignin, hemicellulose and cellulose.Furthermore, the enzymes involved in the degradation of these materials(e.g., cellulase, xylanase, ligninase, glucuronidase,arabinofuranosidase, arabinogalactanase, ferulic acid esterase, lipase,pectinase, glucomannase, amylase, laminarinase, xyloglucanase,galactanase, glucoamylase, pectate lyase, chitinase,exo-β-D-glucosaminidase, cellobiose dehydrogenase, and acetylxylanesterase, xylosidase, α-L-arabinofuranosidase, feruloyl esterase,endoglucanase, β-glucosidase, Mn-peroxidase, and laccase) have beenstudied extensively in F. oxysporum strain F3 (Xiros et al. 2009,Enhanced ethanol production from brewer's spent grain by a Fusariumoxysporum consolidated system. Biotechnol Biofuels. 10:4). Consequently,F. oxysporum strains, including Strain MK7, are expected to be fullyequipped to hydrolyze complex ligno-cellulosic materials such as wheatstraw. It is also expected that since strain MK7 is capable of growth atmuch lower pH (0.7-7.5) in comparison to other Fusarium strains (pH>2;Starkey, 1973), the enzymes for lignin, hemicellulose and cellulosedegradation in strain MK7 will have higher activities at low pH. Enzymeswith high activity under acidic conditions would be especially usefulfor biofuel production when acid hydrolysis is used as a pretreatmentfor ligno-cellulosic materials.

Methods of cloning said enzymes are known in the art. For example, thegene encoding a specific enzyme can be isolated by RT-PCR frompolyadenylated mRNA extracted from fungus, or by PCR from DNA extractedfrom fungus. The resulting product gene can be cloned as a DNA insertinto a vector. The term “vector” refers to the means by which a nucleicacid can be propagated and/or transferred between organisms, cells, orcellular components. Vectors include plasmids, viruses, bacteriophages,pro-viruses, phagemids, transposons, artificial chromosomes, and thelike, that replicate autonomously or can integrate into a chromosome ofa host cell. A vector can also be a naked RNA polynucleotide, a nakedDNA polynucleotide, a polynucleotide composed of both DNA and RNA withinthe same strand, a poly-lysine-conjugated DNA or RNA, apeptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like,that is not autonomously replicating. In many, but not all, commonembodiments, the vectors of the present invention are plasmids orbacmids.

Thus, the invention comprises nucleotides that encode acid pH tolerantenzymes, including chimeric molecules, coned into an expression vectorthat can be expressed in a host cell other than Fusarium oxysporum(e.g., other fungus strain cell, a yeast cell, a bacteria cell, a plantcell, et al.). An “expression vector” is a vector, such as a plasmidthat is capable of promoting expression, as well as replication of anucleic acid incorporated therein. Typically, the nucleic acid to beexpressed is “operably linked” to a promoter and/or enhancer, and issubject to transcription regulatory control by the promoter and/orenhancer. The promoter can be a constitutive expression promoter, aninducible expression promoter, a tissue-specific expression promoter, ora subcellular organelle-specific expression promoter. In one embodiment,nucleotides encoding one or more acid pH tolerant enzymes are expressedin one expression vector transformed into a host cell, or more than oneexpression vectors co-transformed into the host cell.

In one embodiment, the acid pH tolerant enzymes purified from theisolated fungal strain and/or its progeny of the present invention, orproduced in a recombinant host cell, when tested in vitro compared witha homologous enzyme from a non-acid tolerant Fusarium oxysporum strains(e.g., Fusarium strains that can only growth with a pH of at least 3.0,see Starkley et al., Effect of pH on toxcicity of copper to Scytalidiumsp., a copper-tolerant fungus, and some other fungi. J. gen. Microbiol.78: 217-225), has about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, about 100%, about 150%, about 200%, about 250%, about 300%,about 400%, about 500%, about 550%, about 600%, about 650%, about 700%,about 800%, about 850%, about 900%, or about 1000% more activity under apH of less than 3.0, less than 2.9, less than 2.8, less than 2.7, lessthan 2.6, less than 2.5, less than 2.4, less than 2.3, less than 2.2,less than 2.1, less than 2.0, less than 19, less than 1.8, less than1.7, less than 1.6, less than 1.5, less than 1.4, less than 1.3, lessthan 1.2, less than 1.1, less than 1.0, less than 0.9, less than 0.8,less than 0.7, less than 0.6, or less than 0.5.

Thus, the acid pH tolerant enzymes from F. oxysporum strain and/or itsprogeny of the present invention, and nucleotides encoding such enzymescan be isolated and utilized for many purposes. In one embodiment, suchenzymes can be used in the pretreatment of ligno-cellulosic feedstocksfor ethanol production prior to fermentation by other organisms(bacteria, fungi, yeasts, et al.) to degrade lignin, cellulose andhemicelluloses. For example, these enzymes are used right afterhydrolysis of ligno-cellulosic feedstocks (esp. acid hydrolysis),wherein the lignin, cellulose and hemicelluloses are degraded by theenzymes. No neutralization step is required compared to conventionalmethods due to the acid tolerant property of the enzymes derived fromthe isolated fungal strain of the present invention. In one embodiment,the acid-tolerant enzymes produced by the isolated fungal strain of thepresent invention can be used for current biofuel productiontechnologies that involve acid pretreatment and enzymatic degradation.

Toxins Produced by F. oxysporum

Currently, microbial based production of biofuels requires the use ofcostly and time consuming methods to prevent contamination. Strain MK7is highly resistant to contamination by other organisms, because membersof the Fusarium genus are known to generate toxins (e.g., naphthazarinpigments), which have potent antibiotic, insecticidal and phytotoxicproperties. Thus, strain MK7 requires little or non external antibioticsto be added when used in the production of biofuel and/or biofuelprecursors. Fusarium oxysporum in general produces nine differenttoxins, with production depending on their relationship with differenthost plants (Marasas et al., 1984, Toxigenic Fusarium species, identityand mycotoxicology, ISBN 0271003480). The production of toxins alsovaries with media used in vitro fermentation. The toxins produced andsecreted by Fusarium species include, but are not limited to, bikaverin,enniatins, fusaric acid, lycomarasmin, moniliformin, oxysporone,trichothecenes, zearelones, various naphthoquinones and anthraquinones(e.g., nonaketide naphthazarin quinones, bikaverin and norbikaverin,heptaketides, nectriafurone, 5-O-methyljavanicin, and anhydrofusarubinlactol). For example, the toxins from Fusarium species may include,4-Acetoxyscirpenediol(4-3-acetoxy-3,15-dihydroxy-12,13-epoxytrichothec-9-ene. A similarcompound, monodeacetylanguidin=4- or 15-acetylscirpentriol),3-Acetyldeoxynivalenol (Deoxynivalenol monoacetate,3″-acetoxy-7″,15-dihydroxy-12,13-epoxytrichothec-9-en-8-one),8-Acetylneosolaniol (Neosolaniol monoacetate,4″,8″,15-triacetoxy-3″-hydroxy-12,13-epoxytrichothec-9-ene), 4- or15-Acetylscirpentriol (4-Acetoxyscirpenediol), Acetyl T-2 toxin(3″,4″,15-triacetoxy-8″-(3-methylbutyry(oxy)-12,13-epoxytricho-thec-9-ene),Anguidin (Diacetoxyscirpenol), Avenacein, Beauvericin, Butenolide(4-acetamido-4-hydroxy-2-butenoic-acid-lactone), Calonectrin(3″,15-diacetoxy-12,13-epoxytrichothec-9-ene), 15-Deac etylc alonectrin(15-De-0-acetylcalonectrin,3′-acetoxy-15-hydroxy-12,13-epoxytrichothec-9-ene), Deoxynivalenol (Rdtoxin, Vomitoxin,3″,7″,15-trihydroxy-12,13-epoxytricho-thec-9-en-8-one), Deoxynivalenoldiacetate (Diacetyldeoxynivalenol), Deoxynivalenol monoacetate(3-Acetyldeoxynjvalenol), Diacetoxyscirpendiol(7″-Hydroxydiacetoxyscirpenol), Diacetoxyscirpenol (Anguidin,4,15-diacetoxy-3′-hydroxy12,13-epoxytrjchothec-9-ene),Diacetoxyscerpentriol (7″,8″-Dihydroxydiacetoxyscirpenol),Diacetyldeoxynivalenol (Deoxynivalenol diacetate,3″,15-diacetoxy-7-hydroxy 12,13-epoxytrichothec-9-en-8-one),Diacetylnivalenol (Nivalenol diacetate,4,15-diacetoxy-3′,7′-dihydroxy-12,13-epoxytrichothec-9-en-8-one),7″,8″-Dihydroxydiacetoxyscirpenol (Diacetoxyscirpentriol,4,15-diacetoxy-3″,7″,8″-trihydroxy-12,13-epoxytrichothec-9-ene),Enniatins, Fructigenin, Fumonisin B₁ (1,2,3-propanetricarboxylic acid1,-1-[1-(12-amino-4,9,11-trihydroxy-2-methyltridecyl)-2-(1-methylpentyl)-1,2-ethanediyl]ester;macrofusine), Fusarenon (Fusarenon-X, Fusarenon, Monoacetylnivalenol,Nivalenol monoacetate,4-acetoxy-3″,7″,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one) Fusaricacid (Fusarinic acid, 5-butylpicolinic acid), Fusarinic acid (Fusaricacid), F-2 (Zearalenone), HT-2toxin=15-acetoxy-3″,4-dihydroxy-8″-(3-methylbutyryloxy)-12-epoxytricho-thec-9-ene.7″-Hydroxy-diacetoxyscirpenol (Diacetoxyscirpendiol,4,15-diacetoxy-3″,7″-dihydroxy-12,13-epoxytrichothec-9ene),8″-Hydroxydiacetoxyscirpenol (Neosolaniol), 1,4-Ipomeadiol(1-(3-furyI)-1,4-pentanediol), Ipomeanine(1-(3-furyl)-1,4-pentanetione), 1-Ipomeanol(1-(3-furyl)-1-hydroxy-4-pentanone), 4-1pomeanol(1-(3-furyl)-4-hydroxy4pentanone), Lateritin, Lycomarasmin, Moniliformin(potassium or sodium salt of 1-hydroxycyclobut-1-ene-3,4-dione),Monoacetoxyscirpenol(15-acetoxy-3″,4″-dihydroxy-12,13-epoxytrichothec-9ene),Monoacetylnivalenol (Fusarenon-X), Monodeacetylanguidin(4-Acetoxyscirpenediol), Neosolaniol (8″-Hydroxydiacetoxyscirpenol,4,15-diacetoxy-3″8″-dihydroxy-12,13-epoxytrichothec-9-ene),Neosolaniolacetate (8-Acetylneosolaniol), Neosolaniol monoacetate(8-Acetylneosolaniol), Nivalenol(3″,4″,7″,15″-tetrahydroxy-12,13-epoxy-trichothec-9-en-8-one), Nivalenoldiacetate (Diacetylnivalenol), Nivalenol monoacetate (Fusarenon-X), NT-1toxin (T-1 toxin,4″,8″-diacetoxy-337,15-dihydroxy-12,13-epoxy-trichothec-9-ene), NT-2toxin (4″oxy-3″,8″,15-trihydroxy-12,13-epoxytrichothec-9-ene), Rd toxin(Deoxynivalenol), Sambucynin, Scirpentriol(3″,4″,15″-trihydroxy-12,13-epoxytrichothec-9-ene), Solaniol(Neosolaniol), T-1 toxin (NT-1 toxin), T-2 toxin(4″,15″-diacetoxy-3″-hydroxy-8″-(3-methylbutyrlyloxy)-12,13-epoxytrichothec-9-ene),Triacetoxy-scirpendiol(4″,8″,15″-triacetoxy-3″,7″-dihydroxy-12,13-epoxytrichothec-9-ene),Triacetoxy-scirpenol 3″, 4″,15″-triacetoxy-12,13-epoxytrichothec-9-ene),Vomitoxin (Deoxynivalenol), Yavanicin, Zearalenol(2,4-dihydroxy-6-(6,10-dihydroxy-trans-1-undecenyl)-benzoicacid-lactone), Zearalenone(6-(10-hydroxy-6-oxo-trans-1-undecenyl)—resorcylic acid lactone). Moredetailed toxins produced by F. oxysporum are described in Tatum et al.(Naphthoquinones produced by Fusarium oxysporum isolated from citrus.1985, Phytochemistry 24:457-459), Tatum et al. (Naphthofurans producedby Fusarium oxysporum isolated from citrus. 1987, Phytochemistry,26:2499-2500), Baker et al. (Novel anthraquinones from stationarycultures of Fusarium oxysporum. 1998, J Ferment Bioeng 85:359-361).Thrane (Fusarium species on their specific profiles of secondarymetabolites, in Fusarium. Mycotoxins, taxonomy and pathogenicity, 1989,ed by Chelkowski J, Elsevier, N.Y., USA, pp 199-225); Baker et al.,Antimicrobial activity of naphthoquinones from Fusaria, Mycopathologia111: 9-15, 1990; Marasas et al. (Toxigenic Fusarium species, identityand mycotoxicology, 1984, Pennsylvania State University Press,University Park, Pa., USA), each of which is incorporated by referent inits entirety for all purposes.

In another embodiment, the toxins (e.g., naphthazarin pigments) producedby strain MK7 may also be used for other industrial applications. Forexample, the toxins (e.g., naphthazarin pigments) produced by strain MK7can be used as antibiotics, insecticides, and/or herbicides. In oneembodiment, naphthazarin pigments produced by strain MK7 are appliedtogether with a second chemical which is selected from otherantibiotics, insecticides, and/or herbicides.

Siderophores Produced by F. oxysporum

Fusarium oxysporum strains are known to produce siderophores that may beutilized to complex metals for industrial applications. Siderophoresproduced by the isolated fungal strain of the present invention areexpected to be functional at low pH.

Siderophores (Greek: “iron carrier”) are small, high-affinity ironchelating compounds secreted by microorganisms such as bacteria, fungiand grasses (Neilands et al., A Crystalline Organo-iron Pigment from aRust Fungus (Ustilago sphaerogena), 1952. J. Am. Chem. Soc 74:4846-4847, Neilands et al., Siderophores: Structure and Function ofMicrobial Iron Transport Compounds. 1995, J. Biol. Chem. 270:26723-26726). Siderophores are amongst the strongest soluble Fe³⁺binding agents known. Iron is essential for almost all life, essentialfor processes such as respiration and DNA synthesis. Despite being oneof the most abundant elements in the Earth's crust, the bioavailabilityof iron in many environments such as the soil or sea is limited by thevery low solubility of the Fe³⁺ ion. Microbes release siderophores toscavenge iron from these mineral phases by formation of soluble Fe³⁺complexes that can be taken up by active transport mechanisms. Manysiderophores are nonribosomal peptides (Miethke et al., 2007,Siderophore-Based Iron Acquisition and Pathogen Control. Microbiol. Mol.Bio. Reviews. 71: 413-451), although several are biosynthesizedindependently (Challis et al., 2005, A widely distributed bacterialpathway for siderophore biosynthesis independent of nonribosomal peptidesynthetases. Chem Bio Chem, 6:601-611). Siderophores are amongst thestrongest binders to Fe³⁺ known, with enterobactin being one of thestrongest of these. Because of this property, they have attractedinterest from medical science in metal chelation therapy, with thesiderophore desferrioxamine B gaining widespread use in treatments foriron poisoning and thalassemia. Siderophores also can chelate othermetals, which include, but are not limited to, Aluminum, Gallium,Chromium, Copper, Zinc, Lead, Manganese, Cadmium, Vanadium, Indium,Plutonium, and Uranium.

Siderophores usually form a stable, hexadentate, octahedral complex withFe³⁺ preferentially compared to other naturally occurring abundant metalions, although if there are less than six donor atoms water can alsocoordinate. The most effective siderophores are those that have threebidentate ligands per molecule, forming a hexadentate complex andcausing a smaller entropic change than that caused by chelating a singleferric ion with separate ligands. For a representative collection ofsiderophores see Miller et al. (Studies and Syntheses of Siderophores,Microbial Iron Chelators, and Analogs as Potential Drug Delivery Agents.2000, Current Medicinal Chemistry 7: 159-197). Siderophores are usuallyclassified by the ligands used to chelate the ferric iron. The majorgroups of siderophores include the catecholates (phenolates),hydroxamates and carboxylates (e.g. derivatives of citric acid). Citricacid can also act as a siderophore. The wide variety of siderophores maybe due to evolutionary pressures placed on microbes to producestructurally different siderophores which cannot be transported by othermicrobes' active transport systems, or in the case of pathogensdeactivated by the host organism. Non-limiting examples of siderophoresinclude, hydroxamate siderophores (e.g., ferrichrome in Ustilagosphaerogena, Desferrioxamine B (Deferoxamine) in Streptomyces pilosus orStreptomyces coelicolor, Desferrioxamine E in Streptomyces coelicolor,fusarinine C in Fusarium roseum, ornibactin in Burkholderia cepacia),catecholate siderophores (e.g., enterobactin in Escherichia coli andenteric bacteria, bacillibactin in Bacillus subtilis and Bacillusanthracis, vibriobactin in Vibrio cholerae), and mixed ligandssiderophores (e.g., azotobactin in Azotobacter vinelandii, pyoverdine inPseudomonas aeruginosa, yersiniabactin in Yersinia pestis). More fungalsiderophores are described in Renshaw et al. (Fungal siderophores:structures, functions and applications, 2002, Mycol. Res. 106 (10):1123-1142).

Siderophores have applications in medicine for iron and aluminumoverload therapy and antibiotics for better targeting. Understanding themechanistic pathways of siderophores has led to opportunities fordesigning small-molecule inhibitors that block siderophore biosynthesisand therefore bacterial growth and virulence in iron-limitingenvironments (Ferreras et al., 2005, Small-molecule inhibition ofsiderophore biosynthesis in Mycobacterium tuberculosis and Yersiniapestis. Nature Chemical Biology 1: 29-32). Siderophores are useful asdrugs in facilitating iron mobilization in humans, especially in thetreatment of iron diseases, due to their high affinity for iron. Onepotentially powerful application is to use the iron transport abilitiesof siderophores to carry drugs into cells by preparation of conjugatesbetween siderophores and antimicrobial agents. Because microbesrecognize and utilize only certain siderophores, such conjugates areanticipated to have selective antimicrobial activity.

Microbial iron transport (siderophore)-mediated drug delivery makes useof the recognition of siderophores as iron delivery agents in order tohave the microbe assimilate siderophore conjugates with attached drugs.These drugs are lethal to the microbe and cause the microbe to commitsuicide when it assimilates the siderophore conjugate. Through theaddition of the iron-binding functional groups of siderophores intoantibiotics, their potency has been greatly increased. This is due tothe siderophore-mediated iron uptake system of the bacteria.

Thus, in one embodiment, siderophores can be extracted from the isolatedF. oxysporum strain MK7. For example, the strain MK7 can be culturedusing suitable medium as described in the present invention, orligno-cellulosic feedstocks, carbon containing waste products,carbohydrates, or a combination thereof under anaerobic or aerobicconditions. Methods of extracting siderophores are known to one skilledin the art. Siderophore extraction into an organic solvent, ethylacetate in the case of catechol type or either benzyl alcohol orchloroform:phenol (1:1) for the hydroxamate type, have been used as aneffective purification steps for many type of siderophores since bothsalts and macromolecules are removed effectively (Neilands et al.,Microbial iron compounds. 1981, Ann. Rev. Biochem. 50, 715-731). Thepolystyrene resin Amberlite XAD, for the purification of neutral,ferrichrome type of siderophores (Horowitz et al., Isolation andidentification of the conidial growth factor of Neurospora crassa. 1976,J. Bacteria 127, 135-140.) and polyamide resin for chromatographicseparation of catechol type have been widely used.

In addition, the present invention provides methods of detoxifyingfluids containing metal ions and/or recovering metal ions from fluids bybiosorption. In one embodiment, a composition comprising the isolated F.oxysporum strain MK7, or a composition comprising siderophores extractedfrom the isolated F. oxysporum strain MK7, can be used to detoxifyfluids containing metals as described above and/or recover such metals.In one embodiment, a composition comprising sufficient amount ofisolated F. oxysporum strain MK7 or siderophores extracted from theisolated F. oxysporum strain MK7 is mixed with a sample containingmetals for a certain time which allows majority of the metal ions in thefluids bind to siderophores. In one embodiment, such a sample can be awaste fluid containing one or more types of metal ions (e.g., miningwaste fluids, industrial waste fluids, et al.). In one example, thefluids contain at least one metal ion selected from the group consistingof Ag, Zn, Fe, Al, Be, Pb, Cu, Cr, Ni, Cd, Co, Ni, Mn, Pd, Pt, U, Th,Mo, Sn, Ti, As, Au and Hg. In one embodiment, the total initial metalions concentration ranges from about 1 ppm to about 100,000 ppm, byweight. For example, the total metal ions concentration ranges fromabout 0.5 ppm (e.g. Cd) to about 250 ppm (e.g. Zn) depending on themetal constituent. In one embodiment, the waste fluids are mixed withisolated F. oxysporum strain MK7 in a bioreactor, wherein additionalmedium and nutrients for fungal growth are added into the bioreactor toallow proliferation of strain MK7. One of the most relevant requirementsfor the technological application of biosorption is the biomass fixationto an attaching medium in order to allow the biosorbent to be kept in areactor, so it can be reused. This has been performed frequently byimmobilizing the microorganisms on a matrix. Thus, in one embodiment,the strain MK7 or siderophore is immobilized on a matrix. There are manyexamples of the application of these methodologies, the mostrepresentative can be found in the following scientific researches:Brierley, Production and application of a Bacillus-based product for usein metals biosorption. In: B. Volesky, Editor, Biosorption of HeavyMetals, CRC Press, Boca Raton, Fla. (1990), pp. 305-312; Brierley,Immobilization of biomass for industrial application of biosorption. In:A. E. Torma, M. L. Apel and C. L. Brierley, Editors,Biohydrometallurgical Technologies, Proceedings of the InternationalBiohydrometallurgy Symposium, The Minerals, Metals and MaterialsSociety, Warrendale, Pa. (1993), pp. 35-44; Tsezos y Deutschmann (1990).An Investigation of Engineering Parameters for the use of ImmobilizedBiomass Particles in Biosorption. J. Chem. Technol. Biotechnol., 48,29-39; Gilson y Thomas (1995), Calcium alginate bead manufacture: withand without immobilized yeast. Drop formation at a two-fluid nozzle. J.Chem. Technol. Biotechnol. 62 pp. 227-232; Bedell y Damall, (1990),Immobilization of nonviable, biosorbent, algal biomass for the recoveryof metal ions. In: B. Volesky, Editor, Biosorption of Heavy Metals, CRCPress, Boca Raton, Fla., pp. 313-326; Figueira et al. (2000),Biosorption of metals in brown seaweed biomass. Water Res. 34 pp.196-204; Kratochvil et al. (1997) Optimizing Cu removal/recovery in abiosorption column. Water Res. 31 pp. 2327-2339; Kratochvil y Volesky,(2000), Multicomponent biosorption in fixed beds. Water Res. 34 pp.3186-3196; Trujillo et al, (1991), Mathematically modeling the removalof heavy metals from wastewater using immobilized biomass. Environ. Sci.Technol. 25 pp. 1559-1565. The immobilizing agents or the most commonlyused matrixes are alginate, polyacrylamine, polysulfone, silica,cellulose and glutaraldehyde.

Metal ions that bind to biosorbent (strain MK7 or siderophores extractedfrom strain MK7) through biosorption step as described above aresubjected desorption step which allows to elute the metal ions from thesiderophores and regenerate the metals binding capacity of the fungalstrain or extracted siderophores. This is performed by treating themixture of fungal strain and waste fluids or the mixture of extractedsiderophores and waste fluids with an acid solution and, subsequently,with a base solution to neutralize the acid in the mixture. The acid(e.g., concentrated H₂SO₄ 95-97%) solution is added into to the mixturefor desorption, wherein the metal ions are released from the immobilizedbiosorbent (strain MK7 or siderophores extracted from strain MK7). Afterthe desorption step, the mixture is neutralized by using a base solution(e.g., a concentrated sodium hydroxide solution (e.g. NaOH 50%).

Plasticizer

The inventors also discovered that the present isolated F. oxysporumstrain can produce plasticizers, e.g., Diisooctyl phthalate (DIOP) andHexanedioic acid, mono(2-ethylhexyl)ester (a.k.a. 2-Ethylhexyl hydrogenadipate).

Plasticizers (dispersants) are additives that increase the plasticity orfluidity of the material to which they are added; these includeplastics, cement, concrete, wallboard, and clay. Although the samecompounds are often used for both plastics and concretes the desiredeffect is slightly different. The worldwide market for plasticizers in2004 had a total volume of around 5.5 million tons, which led to aturnover of just over 6 billion pounds.

Plasticizers for concrete soften the mix before it hardens, increasingits workability or reducing water, and are usually not intended toaffect the properties of the final product after it hardens.Plasticizers for wallboard increase fluidity of the mix, allowing loweruse of water and thus reducing energy to dry the board. The plasticizersfor plastics soften the final product increasing its flexibility.

Plasticizers for plastics are additives, most commonly phthalates, thatgive hard plastics like PVC the desired flexibility and durability. Theyare often based on esters of polycarboxylic acids with linear orbranched aliphatic alcohols of moderate chain length. Plasticizers workby embedding themselves between the chains of polymers, spacing themapart (increasing the “free volume”), and thus significantly loweringthe glass transition temperature for the plastic and making it softer.For plastics such as PVC, the more plasticizer added, the lower its coldflex temperature will be. This means that it will be more flexible,though its strength and hardness will decrease as a result of it.

Ester plasticizers serve as plasticizers, softeners, extenders, andlubricants, esters play a significant role in rubber manufacturing. Thebasic function of an ester plasticizer is to modify a polymer or resinenhancing its utility. Ester plasticizers make it possible to achieveimproved compound processing characteristics, while also providingflexibility in the end-use product. Ester plasticizers are selectedbased upon cost-performance evaluation. The rubber compounder mustevaluate ester plasticizers for compatibility, processibility,permanence and other performance properties. The wide varieties of esterchemistries that are in production include sebacates, adipates,gluterates, phthalates, azelates, and other specialty blends. This broadproduct line provides an array of performance benefits required for themany elastomer applications such as tubing and hose products, seals andgaskets, belts, wire and cable and print rolls. Low to high polarityesters provide utility in a wide range of elastomers including nitrile,polychloroprene, EPDM, chlorinated polyethylene, and epichlorohydrin.Plasticizer-elastomer interaction is governed by many factors such assolubility parameter, molecular weight and chemical structure.Compatibility and performance attributes are key factors in developing arubber formulation for a particular application.

None-limiting examples of plasticizers include, phthalate-basedplasticizers (e.g., Bis(2-ethylhexyl) phthalate (DEHP), Diisononylphthalate (DINP), Bis(n-butyl)phthalate (DnBP, DBP), Butyl benzylphthalate (BBzP), Diisodecyl phthalate (DIDP), Di-n-octyl phthalate (DOPor DnOP), Diisooctyl phthalate (DIOP), Diethyl phthalate (DEP),Diisobutyl phthalate (DIBP), and Di-n-hexyl phthalate), Trimellitates(e.g., Trimethyl trimellitate (TMTM), Tri-(2-ethylhexyl) trimellitate(TEHTM-MG), Tri-(n-octyl,n-decyl) trimellitate (ATM), Tri-(heptyl,nonyl)trimellitate (LTM), n-octyl trimellitate (OTM)), Adipate-basedplasticizers (Bis(2-ethylhexyl)adipate (DEHA), Dimethyl adipate (DMAD),Monomethyl adipate (MMAD), Dioctyl adipate (DOA)), Dibutyl sebacate(DBS), Dibutyl maleate (DBM), Diisobutyl maleate (DIBM), benzoates,epoxidized vegetable oils, sulfonamides, N-ethyl toluene sulfonamide(o/p ETSA), ortho and para isomers, N-(2-hydroxypropyl) benzenesulfonamide (HP BSA), N-(n-butyl) benzene sulfonamide (BBSA-NBBS),Organophosphates (e.g., Tricresyl phosphate (TCP), Tributyl phosphate(TBP), Glycols/polyethers, Triethylene glycol dihexanoate (3G6, 3GH),Tetraethylene glycol diheptanoate (4G7)), polymeric plasticizers, andpolybutene.

As a plasticizer, DIOP is an all-purpose plasticizer for polyvinylchloride, polyvinyl acetate, rubbers, cellulose plastics, andpolyurethane.

The present invention is further illustrated by the following examplesthat should not be construed as limiting. The contents of allreferences, patents, and published patent applications cited throughoutthis application, as well as the Figures, are incorporated herein byreference in their entirety for all purposes.

EXAMPLE Example 1 Isolation and Growth of Fusarium oxysporum Strain MK7

The isolated fungal Fusarium oxysporum strain MK7 of the presentinvention was isolated from Yellowstone National Park underNPS-permitted project study number YELL-2007-SCI-1976.

Methods of isolating Fusarium oxysporum strain MK7 were previouslydescribed. For example, 0.5 grams of algae-fungal biomass was inoculatedin 200 mL of native filtered spring water from Yellowstone National parkat pH 2.5 and cultured in sunlight for 10 days, after which the algaewere consumed. 50 uL of the remaining pinkish biomass was streaked onto1.2% agar with synthetic media (Kozubal et al., 2008) at pH 3.0.

The isolated fungus was studied and confirmed to be a Fusarium oxysporumstrain by morphological characteristics, which include hyphae that areseptate and hyaline, short and unbranched conidiophores; abundant andtypical Fusarium oxysporum-like sickle-shaped macroconidia with5-septate measuring approximately 25-50×3-4.5 μm; and abundant slightlycurved non-septate microconidia about 5-10×2.0-3.5 μm. In addition,BLAST results of 18S rRNA and ITS (internal transcribed spacer) regionDNA sequences (SEQ ID NO. 1) indicate that the organism is a Fusariumoxysporum strain.

During growth of strain MK7, enzymes were produced that degrade lignin,cellulose and hemicelluloses. The fungus utilizes the resultantdegradation products to generate lipids or produce ethanol and hydrogen,depending on the conditions.

SEQ ID NO. 1 18S rRNA and ITS region DNA sequence ofFusarium oxysporum strain MK7CCGCGGGGAATACTACCTGATCCGAGGTCACATTCAGAGTTGGGGGTTTACGGCTTGGCCGCGCCGCGTACCAGTTGCGAGGGTTTTACTACTACGCAATGGAAGCTGCAGCGAGACCGCCACTAGATTTCGGGGCCGGCTTGCCGCAAGGGCTCGCCGATCCCCAACACCAAACCCGGGGGCTTGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTTGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTTATTTATGGTTTTACTCAGAAGTTACATATAGAAACAGAGTTTAGGGGTCCTCTGGCGGGCCGTCCCGTTTTACCGGGAGCGGGCTGATCCGCCGAGGCAACAATTGGTATGTTCACAGGGGTTTGGGAGTTGTAAACTCGGTAATGATCCCTCCGCAGTTCTCACCTACGGATAGGATCATTACCGAGTTTACAACTCCCAAACCCCTGTGAACATACCCATTGTTGCCTCGGCCGGATCAGCCCGCTCCCGGTTAAAACGGGACGGCCCGCCAGAGTACCCCTAAACTCTGTTTCTATATGTAACTTCTGAGTAAAACCATAAATAAATCAAAACTTTCAACACGCATCTCTTGCTTCTGTCATCGATGAAGAACGCAGCAAAATGCGATAGTCATGTGATTGCACATTCAGTGAATCATCGATCTTGACGCACATTGCGCCTGCAGTATTCTGGCGGTCATGCCTGTTCGAGCGTCATTCAGCCCTCAGCCCTCGGTTGTGTTCGGGATCGGCGAGTCCTGCGCCAGCGACCGGATCAGTGGCGTCTGCCTGCGCCTCCATTGCGGTTAGAGTTAAGCCCTCGCCCACTTG TTTTACGCTAAC

Example 2 Lipids Production by Fusarium oxysporum Strain MK7

Fusarium oxysporum strain MK7 inoculum and liquid media (containingAmmonium nitrate 3.5 g/liter, Calcium chloride-2-H₂O 0.4 g/liter,Magnesium sulphate-7-H₂O 0.30 g/liter, Potassium phosphate mono basic 2g/liter, Manganese sulfate 0.5 g/liter, and trace minerals to finalconcentrations described by Kozubal et al., 2008, incorporated herein byreference in its entirety) can be mixed with ligno-cellosic feedstocks,ligno-cellulosic feedstocks, carbon containing waste products or sugarmonomers in an aerobic system. After a given number of days, theorganism can be harvested and used for lipid extraction. The proprietaryprocess may also be conducted in an anaerobic system in which ethanoland hydrogen gas are produced. FIG. 2 shows fungal biomass of Fusariumoxysporum strain MK7 after 7 days of growth on wheat straw at pH 2.5under aerobic conditions. The fungal mat was ready for lipid extraction.

In a test, different types of feedstocks were mixed with Fusariumoxysporum strain MK7 inoculum and liquid media, including glucose,xylose, alfalfa, avicel, white paper, and wheat straw. After 10 days,fungus biomass was harvested for lipid extraction. Lipids in the fungusbiomass were extracted using the 2:1 chloroform/methanol total lipidextraction method described previously (Bligh and Dyer, A rapid methodof total lipid extraction and purification. 1959, Can. J. Biochem.Physiol., 37, 911-917).

Total yield of extracted lipids from strain MK7 growing in eachdifferent feedstock was determined using gravimetric analyticalprocedure. The yields of fungus growing glucose (pH 2.5), 8% wheat straw(pH 2.5), 4% wheat straw (pH 2.5), and 8% wheat straw (pH 2.5; 25 mM Mn)are displayed in FIG. 3. As it shows, strain MK7 can produce lipids fromglucose or wheat straw, and almost 8% of the initial mass of wheat strawwas converted to lipid after 10 days at pH 2.5. Optical and fluorescentmicroscopy revealed the accumulation of large lipid globules within thecells of strain MK7 (FIG. 4).

The fatty acid composition of lipids extracted from cultures grown onwheat straw at pH 2.5 was determined using gas chromatography. Theresults showed that approximately 44% of the total lipid content wascomprised of acid oleic acid (18:1), a mono-unsaturated fatty acid, andabout 46% palitic (16:0) and stearic acids (18:0), which are saturatedfatty acids (FIG. 5). A high concentration of mono-unsaturated fattyacids such as oleic acid is desirable for production of biodiesel due totheir favorable viscosity and reduced problems with oxidation (Pinzi etal., 2009. The Ideal Vegetable Oil-based Biodiesel Composition: A Reviewof Social, Economical and Technical Implications. Energy Fuels,23(5):2325-2341). In this regard, the lipid profile generated byFusarium strain MK7 is more favorable in comparison to that of otherdescribed fungi (Ya et al. 2008, Lipids of Filamentous Fungi as aMaterial for Producing Biodiesel Fuel. Applied Biochemistry andMicrobiology. 44:523-527; Meng et al., 2009, Biodiesel production fromoleaginous microorganisms. Renewable Energy. 34: 1-5) and microalgae,which produce much higher proportions of polyunsaturated fatty acidsthat are prone to oxidation (Christi, Biodiesel from microalgae. 2007,Biotechnology Advances. 25: 294-306).

Example 3 Ethanol Production by Fusarium oxysporum Strain MK7

Fusarium strain MK7 is capable of producing significant amounts ofethanol under anaerobic and microaerobic conditions using five and sixcarbon sugars. Additionally, it is capable of directly convertingacid-pretreated wheat straw to ethanol.

FIG. 6 summarizes ethanol yields for 4% glucose (w/v) at pH 4.5 and 4%wheat straw (w/v) at pH 3 and 4.5 grown under anaerobic conditions insealed serum bottles. The ethanol yields on glucose were similar tothose found for other Fusarium species grown on glucose (Ruiz et al.,2007. Sugar fermentation by Fusarium oxysporum to produce ethanol. WorldJ Microbiol Biotechnol. 23:259-267), and pH neutralized sugars derivedfrom wheat straw, sweet sorghum stalk and spent brewers grainhydrolysates after alkali pretreatment (Christakopoulos et al., 1991,Direct Ethanol Conversion of Pretreated Straw by Fusarium oxysporum.Bioresource Technology. 35: 297-300; Christakopoulos et al., 1993,Direct conversion of sorghum carbohydrates to ethanol by a mixedmicrobial culture. Bioresource Technology, 45: 89-92; Lezinou et al.,1994, Simultaneous saccharification and fermentation of sweet sorghumcarbohydrates to ethanol in a fed-batch process. Biotechnology Letters.16:983-988; and Xiros et al., 2009, Enhanced ethanol production frombrewer's spent grain by a Fusarium oxysporum consolidated system.Biotechnol Biofuels. 10:4). Ethanol yields at pH 2.5 using wheat strawas a feedstock were low for strain MK7, corresponding to results forother fungal and yeast species grown at pH 4.5-5.5 (Christakopoulos etal., 1989, Direct fermentation of cellulose to ethanol by Fusariumoxysporum. Enzyme Microb Tech. 11:236-239; Christakopoulos et al., 1991,Direct Ethanol Conversion of Pretreated Straw by Fusarium oxysporum.Bioresource Technology. 35: 297-300). This is likely due to theinhibition of ethanol fermentation by phenolics, furfurals and othercompounds that are produced during acid hydrolysis (Palmqvist andHahn-Hagerdal, 2000).

Example 4 Hydrogen Production by Fusarium oxysporum Strain MK7

In addition to the production of ethanol during fermentation, strain MK7produces hydrogen gas when grown anaerobically in sealed serum bottlesusing ligno-cellulosic feedstocks. Although no hydrogen was producedduring glucose fermentation, concentrations as high as 4% H₂ gas weremeasured in the gas headspace when wheat straw was used as a growthsubstrate. The presence of a hyd3-like hydrogenase gene sequence, likelyimportant for H₂ production, was confirmed in Strain MK7 usingpolymerase chain reaction.

Example 5 Biofuel or Biofuel Precursors Production by Fusarium oxysporumStrain MK7 with Pretreatments

Various pretreatments may be utilized to enhance conversion rates bystrain MK7 in the production of biofuel or biofuel precursors.Pretreatments may include acidification to less than pH 3.0, Mnaddition, and nutrient addition (e.g. ammonium and phosphate).

Previous work has shown that Mn(III) likely increases the degradation oflignin through the oxidization of lignin phenolic groups coupled withthe reduction of Mn(III) to Mn(II) (Kerem and Hadar, 1995, Effect ofmanganese on preferential degradation of lignin by Pleurotus ostreatusduring solid-state fermentation. Appl Environ Microbiol. 61(8):3057-3062; Boominathan et al., 1992, cAMP-mediated differentialregulation of lignin peroxidase and manganese-dependent peroxi-daseproduction in the white-rot basidiomycete Phanerochaete chrysosporium.Proc. Natl. Acad. Sci. 89:5586-5590; Tebo et al., 1997,Bacterially-mediated mineral formation: Insights into manganese(II)oxidation from molecular genetic and biochemical studies. In: J. F.Banfield and K. H. Nealson (Eds.) Geomicrobiology: Interactions BetweenMicrobes and Minerals. Reviews in Mineralogy. 35:225-266). Oxidation ofMn(II) to Mn(III) by a variety of enzymes can regenerate Mn(III).

Experiments with strain MK7 indicated that the fungus can tolerate veryhigh levels of Mn and that Mn concentrations of 5, 10 and 25 mMincreased wheat straw degradation and fungal growth. Accurate biomassquantification has not yet been completed but visual estimates indicatedan increase in fungal biomass of approximately 10-25%. Lipid productionappears to be enhanced as well.

Strain MK7 also tolerates very acidic pH conditions. To increase theconversion rates by strain MK7 in the production of biofuel or biofuelprecursors using ligno-cellulosic feedstocks, acidification materialsare added into the mixture of strain MK7 inoculum and ligno-cellulosicfeedstocks, wherein the pH of the mixture is less than 3.0, less than2.9, less than 2.8, less than 2.7, less than 2.6, less than 2.5, lessthan 2.4, less than 2.3, less than 2.2, less than 2.1, less than 2.0,less than 19, less than 1.8, less than 1.7, less than 1.6, less than1.5, less than 1.4, less than 1.3, less than 1.2, less than 1.1, lessthan 1.0, less than 0.9, less than 0.8, less than 0.7, less than 0.6, orless than 0.5. A mixture of strain MK7 inoculum and ligno-cellulosicfeedstocks with a pH of 7.0 is included as a control. After incubationfor a proper time, the biofuel or the biofuel precursors are extractedfrom fungal biomass and/or the mixture of fugal biomass and feedstocks.Compared to the control mixture, the acidified mixture of strain MK7inoculum and ligno-cellulosic feedstocks produce about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95%, about 100%, about 150%,about 200%, about 250%, about 300%, about 400%, about 500%, about 550%,about 600%, about 650%, about 700%, about 800%, about 850%, about 900%,or about 1000% more biofuel or biofuel precursors.

Example 6 Enzymes and Antibiotics Production by Fusarium oxysporumStrain MK7

Enzymes (e.g., cellulase, xylanase, ligninase, glucuronidase,arabinofuranosidase, arabinogalactanase, ferulic acid esterase, lipase,pectinase, glucomannase, amylase, laminarinase, xyloglucanase,galactanase, glucoamylase, pectate lyase, chitinase,exo-β-D-glucosaminidase, cellobiose dehydrogenase, and acetylxylanesterase, xylosidase, α-L-arabinofuranosidase, feruloyl esterase,endoglucanase, β-glucosidase, Mn-peroxidase, and laccase) in Fusariumoxysporum strain MK7 are potentially acid pH tolerant. The enzymes, andthe nucleotides encoding the enzymes, are very useful in biotechnology,and thus can be isolated.

To isolate acid tolerant enzymes from F. oxysporum strain MK7,traditional molecular cloning procedures can be applied. For example,the gene encoding a specific enzyme can be isolated by RT-PCR frompolyadenylated mRNA extracted from fungus, or by PCR from DNA extractedfrom fungus. Primers specific for a gene encoding an enzyme of interestare designed based on the genome information of Fusarium oxysporum, orpeptide sequence of a purified enzyme from Fusarium oxysporum MK7, orpeptide sequence of homologous enzymes from Fusarium species. Theresulting product gene can be cloned as a DNA insert into a vector. Thevector can be an expression vector suitable for a host cell (e.g., afungal cell, a bacteria cell, a yeast cell, a plant cell, an insectcell, or an animal cell).

The acid pH tolerant enzymes purified from Fusarium oxysporum MK7, orproduced in a recombinant host cell, when tested in vitro compared witha homologous enzyme from a non-acid tolerant Fusarium oxysporum species,has about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,about 100%, about 150%, about 200%, about 250%, about 300%, about 400%,about 500%, about 550%, about 600%, about 650%, about 700%, about 800%,about 850%, about 900%, or about 1000% more activity under a pH of lessthan 3.0, less than 2.9, less than 2.8, less than 2.7, less than 2.6,less than 2.5, less than 2.4, less than 2.3, less than 2.2, less than2.1, less than 2.0, less than 19, less than 1.8, less than 1.7, lessthan 1.6, less than 1.5, less than 1.4, less than 1.3, less than 1.2,less than 1.1, less than 1.0, less than 0.9, less than 0.8, less than0.7, less than 0.6, or less than 0.5.

Example 7 Lipid Profiles Produced by Strain MK7

The production of total intracellular lipids was determined using directtransesterification coupled with GC-MS analysis as described in Lohmanet al. (2013). Strain MK7 lipid profiles were remarkably consistentamong treatments (i.e. pH, temperature, growth substrate, cultivationduration, moisture content) and were dominated by C16:0 and C18:0, C18:1and C18:2 triacylglycerides (>95% of total lipids, FIG. 7B). Total fuelpotential and extractable lipid fractions show profiles ideal forbiodiesel (FIG. 7A). Fatty acid profiles also show a number of highvalue products including the omega-7 vaccenic acid (Methyl11-octadecenoate), omega-7 palmitoleic acid (methyl hexadec-9-enoate;trade name Provinal™) and tetracosanoic acid, methyl ester (Table 1).These are rare fatty acids not typically found in vegetable oils and mayproduce significantly more revenue per ton of feedstock than biodieselalone.

TABLE 1 Identities, concentrations and prices for lab grade puritycompounds identified by GC-MS. The listed percentage of total lipid foreach compound was produced by strain MK7. Average % Total Price - labName Formula Lipid grade ($/g) Methyl tetradecanoate C₁₅H₃₀O₂ 0.28 23Methyl hexadec-9-enoate C₁₇H₃₂O₂ 0.71 394 (omega-7 palmitoleic acid16:1) Hexadecanoic acid, methyl ester C₁₇H₃₄O₂ 29.3 30 (palmitic acid16:0) C18:1-3 (combination of oleic; C₁₉H₃₄O₂ 50.14 25-900 linoleicacid; conjugated linoleics and linolinec acid) Methyl 11-octadecenoateC₁₉H₃₆O₂ 2.92 945 (omega-7 vaccenic acid; 18:1) Octadecanoic acid,methyl ester C₁₉H₃₈O₂ 14.03 24 (stearic 18:0) Eicosanoic acid, methylester C₂₁H₄₂O₂ 0.59 271 Docosanoic acid, methyl ester C₂₃H₄₆O₂ 0.43 71Tetracosanoic acid, methyl ester C₂₅H₅₀O₂ 0.76 340

FIG. 7A shows the average of total fatty acid methyl esters (FAME) indirect transesterfication (total fuel potential) and extractable lipidfractions as a function of media C:N ratio (n=3). Bars within theextractable lipid fraction bar represent tri-, di- and mono-acylglycerides (TAG, DAG, MAG) and free fatty acids (FFA) components. Insetshows a GC-FID chromatogram with TAG molecules dominating the lipidfraction. FIG. 7B shows FAME profile of lipids generated from directtransesterification of all fatty acids (Direct) to FAME, and FAMEderived from only extractable lipid precursors (Extractable). Insetshows GC-MS chromatograms for the Direct and Extractable fractions.

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materials,similar or equivalent to those described herein, can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications, patents, and patentpublications cited are incorporated by reference herein in theirentirety for all purposes.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

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1. A method of producing an energy-rich substrate, said methodcomprising contacting a carbon source with an isolated Fusariumoxysporum strain to form a mixture, incubating said mixture for a periodof time, and obtaining the energy-rich substrate following such contact.2. The method of claim 1, wherein the Fusarium oxysporum strain is anMK7 strain, for which a representative sample has been deposited as ATCCAccession Deposit No. PTA-10698.
 3. The method of claim 2, wherein thecarbon source is a biomass product.
 4. The method of claim 2, whereinthe carbon source is a cellulosic biomass product.
 5. The method ofclaim 2, wherein the carbon source is a ligno-cellulosic feedstock orlingo-cellulosic waste.
 6. The method of claim 2, wherein the carbonsource is a carbohydrate.
 7. The method of claim 2, wherein the Fusariumoxysporum strain and the carbon source are incubated in anaerobic ormicroaerobic conditions.
 8. The method of claim 7, wherein the resultingenergy-rich substrate is selected from the group consisting of ethanoland hydrogen gas.
 9. The method of claim 2, comprising a pretreatmentstep, wherein said pretreatment step is selected from the groupconsisting of: reducing the pH of the carbon source and Fusariumoxysporum mixture, adding manganese to the carbon source and Fusariumoxysporum mixture, and adding a nutrient to the carbon source andFusarium oxysporum mixture before the incubation step.
 10. The method ofclaim 2, wherein the energy-rich substrate is a lipid, ethanol and/orhydrogen.
 11. The method of claim 2, wherein the energy-rich substrateis a lipid.
 12. A method of producing one or more energy-richmetabolites using a Fusarium oxysporum strain, said method comprisingthe steps of: a) making a mixture of a Fusarium oxysporum MK7 strain,and/or its progeny, with a feedstock material selected from the groupconsisting of ligno-cellulosic feedstocks, carbon containing wasteproducts, carbohydrates, and a combination thereof in a container,wherein the material can support the growth of said MK7 strain and/orits progeny; b) growing said MK7 strain in said mixture to produce oneor more energy-rich metabolites; and c) optionally, isolating said oneor more energy-rich metabolites from the mixture; wherein arepresentative sample of said MK7 strain has been deposited as ATCCAccession Deposit No. PTA-10698.
 13. The method of claim 12, wherein thefeedstock material is wheat straw.
 14. The method of claim 12, whereinthe energy-rich metabolite is ethanol.
 15. The method of claim 12,wherein the energy-rich metabolite is hydrogen gas.
 16. The method ofclaim 12, wherein the energy-rich metabolite is a fatty acid methylester (FAME).
 17. The method of claim 16, wherein the FAME is selectedfrom the group consisting of methyl tetradecanoate, methylhexadec-9-enoate, hexadecanoic acid, methyl ester, oleic acid, linoleicacid, conjugated linoleics, linolinec acid, methyl 11-octadecenoate,Octadecanoic acid, methyl ester, eicosanoic acid, methyl ester,docosanoic acid, methyl ester, and tetracosanoic acid, methyl ester. 18.The method of claim 16, wherein the FAME is Omega-7 vaccenic acid. 19.The method of claim 12, wherein the mixture is incubated in anaerobic ormicroaerobic conditions.
 20. The method of claim 12, further comprisinga pretreatment step, wherein said pretreatment step is selected from thegroup consisting of: reducing the pH of the mixture, adding manganese tothe mixture, and adding a nutrient to the mixture.